Hla-based methods and compositions and uses thereof

ABSTRACT

The present disclosure provides compositions and methods for isolating HLA-peptides from cells. The present disclosure provides a universal platform and methods for profiling the HLA-peptidome, enabling identification of endogenously presented HLA-peptides from cell lines expressing any possible class I or II construct.

CROSS-REFERENCE

This application claims priority to U.S. Provisional Application No.62/457,978, filed Feb. 12, 2017, and U.S. Provisional Application No.62/461,162, filed Feb. 20, 2017, each of which is incorporated herein byreference in their entirety.

BACKGROUND

The major histocompatibility complex (MHC) is a gene complex encodinghuman leukocyte antigen (HLA) genes. HLA genes are expressed as proteinheterodimers that are displayed on the surface of human cells tocirculating T cells. HLA genes are highly polymorphic, allowing them tofine-tune the adaptive immune system. Adaptive immune responses rely, inpart, on the ability of T cells to identify and eliminate cells thatdisplay disease-associated peptide antigens bound to human leukocyteantigen (HLA) heterodimers.

In humans, endogenous and exogenous proteins can be processed intopeptides by the proteasome and by cytosolic and endosomal/lysosomalproteases and peptidases and presented by two classes of cell surfaceproteins encoded by MHC. These cell surface proteins are referred to ashuman leukocyte antigens (HLA class I and class II), and the group ofpeptides that bind them and elicit immune responses are termed HLAepitopes. HLA epitopes are a key component that enables the immunesystem to detect danger signals, such as pathogen infection andtransformation of self. Circulating CD8+ T cells recognize class I MHC(HLA-A, HLA-B, and HLA-C) epitopes derived from endogenous processingpathways and displayed on almost all nucleated cells. CD4+ T cellsrecognize class II MHC (HLA-DR, HLA-DQ, and HLA-DP) epitopes displayedon antigen presenting cells (APCs), such as dendritic cells andmacrophages. HLA class II-peptide presentation activates helper T cells,subsequently promoting B cell differentiation and antibody production aswell as CTL responses. Activated helper T cells also secrete cytokinesand chemokines that activate and induce differentiation of other Tcells.

The genes coding for HLA heterodimers are highly polymorphic, with morethan 12,000 class I and 4,000 class II allele variants identified acrossthe human population. From maternal and paternal HLA haplotypes, anindividual can inherit different alleles for each of the class I andclass II HLA loci. Class I HLA molecules are heterodimers made up of aheavy α-chain, encoded by class I HLA genes, and the β-2-microglobulin(B2M). Class II HLA molecules are α- and β-chain heterodimers, bothencoded by the class II HLA genes. Because of the α- and β-chain pairingcombinations, the population of HLA heterodimers is highly complex. Inaddition, each HLA heterodimer is estimated to bind thousands ofpeptides with allele-specific binding preferences. In fact, each HLAallele is estimated to bind and present ˜1,000-10,000 unique peptides toT cells; ≤0.1% of ˜10 million potential 9mer peptides from humanprotein-coding genes. Given such diversity in HLA binding, accurateprediction of whether a peptide is likely to bind to a specific HLAallele is highly challenging. Less is known about allele-specificpeptide-binding characteristics of HLA class II molecules because of theheterogeneity of α and β chain pairing, complexity of data limiting theability to confidently assign core binding epitopes, and the lack ofimmunoprecipitation grade, allele-specific antibodies required forhigh-resolution biochemical analyses. Furthermore, analyzing peptideepitopes derived from a given HLA allele raises ambiguity when multipleHLA alleles are presented on a cell surface.

Understanding the binding preferences of every HLA heterodimer is a keyto successfully predicting which neoantigens are likely to elicittumor-specific T cell responses. Clearly, there is a need for methods ofidentifying and isolating specific class I and class II HLA-associatedpeptides (e.g., neoantigen peptides). Such methodology and isolatedmolecules are useful, e.g., for the research of HLA-associated peptides,as well as for the development of therapeutics, including but notlimited to, immune based therapeutics.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.

SUMMARY

The methods and compositions described herein find uses in a wide rangeof applications. For example, the methods and compositions describedherein be used to identify immunogenic antigen peptides and can be usedto develop drugs, such as personalized medicine drugs.

Provided herein is a method of characterizing HLA-peptide complexescomprising: providing a population of cells, wherein one or more cellsof the population of cells comprise a polynucleic acid comprising asequence encoding an affinity acceptor tagged class I or class II HLAallele, wherein the sequence encoding an affinity acceptor tagged HLAcomprises a sequence encoding a recombinant class I or class II HLAallele operatively linked to a sequence encoding an affinity acceptorpeptide; expressing the affinity acceptor tagged HLA in at least onecell of the one or more cells of the population of cells, therebyforming affinity acceptor tagged HLA-peptide complexes in the at leastone cell; enriching for the affinity acceptor tagged HLA-peptidecomplexes; and characterizing HLA-peptide complexes. In someembodiments, the encoded affinity acceptor tagged class I or class IIHLA allele is a soluble affinity acceptor tagged class I or class II HLAallele.

In some embodiments, the characterizing comprises characterizing apeptide bound to the affinity acceptor tagged HLA-peptide complex fromthe enriching. In some embodiments, the method comprises carrying outthe steps of the method for two or more class I and/or class II HLAalleles. In some embodiments, the two or more class I and/or class IIHLA alleles comprise at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50 class I and/or classII HLA alleles. In some embodiments, the affinity acceptor taggedHLA-peptide complexes comprise a transmembrane domain. In someembodiments, the affinity acceptor tagged HLA-peptide complexes comprisean intracellular domain. In some embodiments, the affinity acceptortagged HLA-peptide complexes are not secreted. In some embodiments, theaffinity acceptor tagged HLA-peptide complexes incorporate into a cellmembrane when expressed. In some embodiments, the affinity acceptortagged HLA-peptide complexes are soluble affinity acceptor taggedHLA-peptide complexes. In some embodiments, the affinity acceptor taggedHLA-peptide complexes are not soluble affinity acceptor taggedHLA-peptide complexes. In some embodiments, the method further comprisesgenerating an HLA-allele specific peptide database. In some embodiments,the recombinant class I or class II HLA allele is a single recombinantclass I or class II HLA allele.

In some embodiments, the method comprises: providing a population ofcells each comprising one or more cells comprising an affinity acceptortagged HLA, wherein the affinity acceptor tagged HLA comprises adifferent recombinant polypeptide encoded by a different HLA alleleoperatively linked to an affinity acceptor peptide; enriching foraffinity acceptor tagged HLA-peptide complexes; and characterizing apeptide or a portion thereof bound to the affinity acceptor taggedHLA-peptide complex from the enriching.

In some embodiments, the method comprises introducing one or morepeptides to the population of cells. In some embodiments, theintroducing comprises contacting the population of cells with the one ormore peptides or expressing the one or more peptides in the populationof cells. In some embodiments, the introducing comprises contacting thepopulation of cells with one or more nucleic acids encoding the one ormore peptides. In some embodiments, the one or more nucleic acidsencoding the one or more peptides is DNA. In some embodiments, the oneor more nucleic acids encoding the one or more peptides is RNA,optionally wherein the RNA is mRNA. In some embodiments, the enrichingdoes not comprise use of a tetramer reagent.

In some embodiments, the characterizing comprises determining thesequence of a peptide or a portion thereof bound to the affinityacceptor tagged HLA-peptide complex from the enriching, optionallydetermining whether a peptide or a portion thereof is modified. In someembodiments, the determining comprises biochemical analysis, massspectrometry analysis, MS analysis, MS/MS analysis, LC-MS/MS analysis,or a combination thereof. In some embodiments, the characterizingcomprises evaluating a binding affinity or stability of a peptide or aportion thereof bound to the affinity acceptor tagged HLA-peptidecomplex from the enriching. In some embodiments, the characterizingcomprises determining whether a peptide or a portion thereof bound tothe affinity acceptor tagged HLA-peptide complex from the enrichingcontains one or more mutations. In some embodiments, the characterizingcomprises evaluating associations of peptides with HLA molecules in theaffinity acceptor tagged HLA-peptide complexes.

In some embodiments, the method comprises expressing a library ofpeptides in the population of cells, thereby forming a library ofaffinity acceptor tagged HLA-peptide complexes. In some embodiments, themethod comprises contacting to the population of cells a library ofpeptides or a library of sequences encoding peptides, thereby forming alibrary of affinity acceptor tagged HLA-peptide complexes. In someembodiments, the library comprises a library of peptides associated witha disease or condition. In some embodiments, the library comprises alibrary of peptides derived from a polypeptide drug, such as a biologic(e.g., an antibody drug).

In some embodiments, the disease or condition is cancer, an infectionwith an infectious agent, or an autoimmune reaction. In someembodiments, the method comprises introducing the infectious agent orportions thereof into one or more cells of the population of cells. Insome embodiments, the method comprises introducing a polypeptide drug,such as a biologic (e.g., an antibody drug) or portions thereof into oneor more cells of the population of cells. In some embodiments, themethod comprises characterizing one or more peptides from theHLA-peptide complexes, optionally wherein the peptides are from one ormore target proteins of the infectious agent or the polypeptide drug. Insome embodiments, the method comprises characterizing one or moreregions of the peptides from the one or more target proteins of theinfectious agent or the polypeptide drug.

In some embodiments, the method comprises identifying peptides from theHLA-peptide complexes derived from an infectious agent. In someembodiments, the population of cells is from a biological sample from asubject with a disease or condition. In some embodiments, the populationof cells is a cell line. In some embodiments, the population of cells isa population of primary cells. In some embodiments, the recombinantclass I or class II HLA allele is matched to a subject with a disease orcondition.

In some embodiments, the peptide from the affinity acceptor taggedHLA-peptide complex is capable of activating a T cell from a subjectwhen presented by an antigen presenting cell. In some embodiments, thecharacterizing comprises comparing HLA-peptide complexes from cancercells to HLA-peptide complexes from non-cancer cells. In someembodiments, the population of cells comprises a plurality ofpopulations of cells, each population of cells expressing a differentrecombinant class I or class II HLA allele. In some embodiments, eachpopulation of cells of the plurality is in a same or a separatecontainer.

In some embodiments, the method further comprises isolating peptidesfrom the affinity acceptor tagged HLA-peptide complexes before thecharacterizing. In some embodiments, an HLA-peptide complex is isolatedusing an anti-HLA antibody. In some cases, an HLA-peptide complex withor without an affinity tag is isolated using an anti-HLA antibody. Insome cases, a soluble HLA (sHLA) with or without an affinity tag isisolated from media of a cell culture. In some cases, a soluble HLA(sHLA) with or without an affinity tag is isolated using an anti-HLAantibody. For example, an HLA, such as a soluble HLA (sHLA) with orwithout an affinity tag, can be isolated using a bead or columncontaining an anti-HLA antibody. In some embodiments, the peptides areisolated using anti-HLA antibodies. In some cases, a soluble HLA (sHLA)with or without an affinity tag is isolated using an anti-HLA antibody.In some cases, a soluble HLA (sHLA) with or without an affinity tag isisolated using a column containing an anti-HLA antibody. In someembodiments, the method further comprises removing one or more aminoacids from a terminus of a peptide bound to an affinity acceptor taggedHLA-peptide complex.

In some embodiments, the population of cells is a population of low cellsurface HLA class I or class II expressing cells. In some embodiments,the population of cells expresses one or more endogenous HLA alleles. Insome embodiments, the population of cells is an engineered population ofcells lacking one or more endogenous HLA class I alleles. In someembodiments, the population of cells is an engineered population ofcells lacking endogenous HLA class I alleles. In some embodiments, thepopulation of cells is an engineered population of cells lacking one ormore endogenous HLA class II alleles. In some embodiments, thepopulation of cells is an engineered population of cells lackingendogenous HLA class II alleles. In some embodiments, the population ofcells is an engineered population of cells lacking endogenous HLA classI alleles and endogenous HLA class II alleles. In some embodiments, thepopulation of cells is a knock-out of one or more HLA class I alleles.In some embodiments, the population of cells is a knock-out of one ormore HLA class II alleles. In some embodiments, the population of cellsis a knock-out of all HLA class I alleles. In some embodiments, thepopulation of cells is a knock-out of all HLA class II alleles. In someembodiments, the population of cells is a knock-out of all HLA class Ialleles and a knock-out of all HLA class II alleles. In someembodiments, the sequence encoding the recombinant class I or class IIHLA allele encodes a class I HLA. In some embodiments, the class I HLAis selected from the group consisting of HLA-A, HLA-B, HLA-C, HLA-E,HLA-F, and HLA-G. In some embodiments, the sequence encoding therecombinant class I or class II HLA allele encodes a class II HLA. Insome embodiments, the class II HLA is selected from the group consistingof HLA-DR, HLA-DQ, and HLA-DP. In some embodiments, the class II HLAcomprises a HLA class II α-chain, a HLA class II β-chain, or acombination thereof. In some embodiments, each sequence encodes at leasttwo different class I and/or class II HLA alleles.

In some embodiments, the at least two different class I and/or class IIHLA alleles are each operatively linked to a sequence encoding anaffinity acceptor peptide. In some embodiments, the at least twodifferent class I and/or class II HLA alleles are each operativelylinked to a sequence encoding a different affinity acceptor peptide. Insome embodiments, the at least two different class I and/or class II HLAalleles are each operatively linked to a sequence encoding an affinityacceptor peptide. In some embodiments, one or more of the at least twodifferent class I and/or class II HLA alleles is operatively linked to asequence encoding a first affinity acceptor peptide and one or more ofthe at least two different class I and/or class II HLA alleles isoperatively linked to a sequence encoding a second affinity acceptorpeptide. In some embodiments, the at least two different class I and/orclass II HLA alleles are each operatively linked to a sequence encodinga different affinity acceptor peptide. In some embodiments, each of theat least two different class I and/or class II HLA alleles are eachoperatively linked to a sequence encoding a different affinity acceptorpeptide. In some embodiments, the at least two different class I and/orclass II HLA alleles are each operatively linked to a sequence encodingan affinity tag. In some embodiments, the method comprises administeringat least a second polynucleic acid comprising a sequence encoding adifferent recombinant HLA allele operatively linked to the same or adifferent affinity acceptor peptide.

In some embodiments, the sequence encoding the affinity acceptor peptideis operatively linked to a sequence that encodes an extracellularportion of the recombinant class I or class II HLA allele. In someembodiments, the encoded affinity acceptor peptide is expressedextracellularly. In some embodiments, the encoded affinity acceptorpeptide is located on an extracellular site of the recombinant class Ior class II HLA allele. In some embodiments, the sequence encoding theaffinity acceptor peptide is operatively linked to the N-terminus of thesequence encoding the recombinant class I or class II HLA allele. Insome embodiments, the sequence encoding the affinity acceptor peptide isoperatively linked to a sequence that encodes an intracellular portionof the recombinant class I or class II HLA allele. In some embodiments,the encoded affinity acceptor peptide is expressed intracellularly. Insome embodiments, the sequence encoding the affinity acceptor peptide isoperatively linked to the C-terminus of the sequence encoding therecombinant class I or class II HLA allele. In some embodiments, thesequence encoding the affinity acceptor peptide is operatively linked toan internal sequence of the sequence encoding the recombinant class I orclass II HLA allele, such as a flexible loop sequence. In someembodiments, the sequence encoding the affinity acceptor peptide isoperatively linked to the sequence encoding the recombinant class I orclass II HLA allele by a linker. In some embodiments, enrichingcomprises enriching for intact cells expressing the affinity acceptortagged HLA-peptide complexes. In some embodiments, the method does notcomprise lysing the cells before enriching. In some embodiments, themethod further comprises lysing the one or more cells before enriching.In some embodiments, enriching comprises contacting an affinity acceptorpeptide binding molecule to the affinity acceptor tagged HLA-peptidecomplexes, wherein the affinity acceptor peptide binding molecule bindsspecifically to the affinity acceptor peptide.

In some embodiments, the affinity acceptor peptide comprises a tagsequence comprising a biotin acceptor peptide (BAP), poly-histidine tag,poly-histidine-glycine tag, poly-arginine tag, poly-aspartate tag,poly-cysteine tag, poly-phenylalanine, c-myc tag, Herpes simplex virusglycoprotein D (gD) tag, FLAG tag, KT3 epitope tag, tubulin epitope tag,T7 gene 10 protein peptide tag, streptavidin tag, streptavidin bindingpeptide (SPB) tag, Strep-tag, Strep-tag II, albumin-binding protein(ABP) tag, alkaline phosphatase (AP) tag, bluetongue virus tag (B-tag),calmodulin binding peptide (CBP) tag, chloramphenicol acetyl transferase(CAT) tag, choline-binding domain (CBD) tag, chitin binding domain (CBD)tag, cellulose binding domain (CBP) tag, dihydrofolate reductase (DHFR)tag, galactose-binding protein (GBP) tag, maltose binding protein (MBP),glutathione-S-transferase (GST), Glu-Glu (EE) tag, human influenzahemagglutinin (HA) tag, horseradish peroxidase (HRP) tag, NE-tag, HSVtag, ketosteroid isomerase (KSI) tag, KT3 tag, LacZ tag, luciferase tag,NusA tag, PDZ domain tag, AviTag, Calmodulin-tag, E-tag, S-tag, SBP-tag,Softag 1, Softag 3, TC tag, VSV-tag, Xpress tag, Isopeptag, SpyTag,SnoopTag, Profinity eXact tag, Protein C tag, S1-tag, S-tag,biotin-carboxy carrier protein (BCCP) tag, green fluorescent protein(GFP) tag, small ubiquitin-like modifier (SUMO) tag, tandem affinitypurification (TAP) tag, HaloTag, Nus-tag, Thioredoxin-tag, Fc-tag, CYDtag, HPC tag, TrpE tag, ubiquitin tag, VSV-G epitope tag, V5 tag,sortase tag, a tag the forms a covalent peptide bond to a bead, or acombination thereof; optionally, wherein the affinity acceptor peptidecomprises two or more repeats of a tag sequence.

In some embodiments, the affinity acceptor peptide binding molecule isbiotin or an antibody specific to the affinity acceptor peptide. In someembodiments, the enriching comprises contacting an affinity molecule tothe affinity acceptor tagged HLA-peptide complexes, wherein the affinitymolecule binds specifically to the affinity acceptor peptide bindingmolecule.

In some embodiments, the affinity molecule comprises a molecule thatbinds to biotin. For example, the affinity molecule can comprisestreptavidin, NeutrAvidin, including protein homologs from otherorganisms and derivatives thereof.

In some embodiments, enriching comprises immunoprecipitating affinityacceptor tagged HLA-peptide complexes. In some embodiments, the affinityacceptor peptide binding molecule is attached to a solid surface. Insome embodiments, the affinity molecule is attached to a solid surface.In some embodiments, the solid surface is a bead. In some embodiments,enriching comprises immunoprecipitating affinity acceptor taggedHLA-peptide complexes with an affinity acceptor peptide binding moleculethat binds specifically to the affinity acceptor peptide.

In some embodiments, the affinity acceptor peptide binding molecule doesnot specifically interact with the amino acid sequence of the encodedrecombinant class I or class II HLA. In some embodiments, enrichingcomprises contacting an affinity molecule specific to an extracellularportion of the recombinant class I or class II HLA allele. In someembodiments, enriching comprises contacting an affinity moleculespecific to an N-terminal portion of the recombinant class I or class IIHLA allele.

In some embodiments, providing comprises contacting the population ofcells with the polynucleic acid. In some embodiments, contactingcomprises transfecting or transducing. In some embodiments, providingcomprises contacting the population of cells with a vector comprisingthe polynucleic acid. In some embodiments, the vector is a viral vector.In some embodiments, the polynucleic acid is stably integrated into thegenome of the population of cells.

In some embodiments, the sequence encoding the recombinant class I orclass II HLA comprises a sequence encoding a HLA class I α-chain. Insome embodiments, the method further comprises expressing a sequenceencoding β2 microglobulin in the one or more cells. In some embodiments,the sequence encoding β2 microglobulin is connected to the sequenceencoding the HLA class I α-chain. In some embodiments, the sequenceencoding β2 microglobulin is connected to the sequence encoding the HLAclass I α-chain by a linker. In some embodiments, the sequence encodingβ2 microglobulin is connected to a sequence encoding a second affinityacceptor peptide. In some embodiments, the sequence encoding therecombinant class I or class II HLA comprises a sequence encoding a HLAclass II α-chain. In some embodiments, the method further comprisesexpressing a sequence encoding a HLA class II β-chain in the one or morecells. In some embodiments, the sequence encoding the HLA class IIβ-chain is connected to the sequence encoding the HLA class II α-chain.In some embodiments, the sequence encoding the HLA class II β-chain isconnected to the sequence encoding the HLA class II α-chain by a linker.In some embodiments, the sequence encoding the HLA class II β-chain isconnected to a sequence encoding a second affinity acceptor peptide.

In some embodiments, the second affinity acceptor peptide is differentthan the first affinity acceptor peptide and is selected from the groupconsisting of biotin acceptor peptide (BAP), poly-histidine tag,poly-histidine-glycine tag, poly-arginine tag, poly-aspartate tag,poly-cysteine tag, poly-phenylalanine, c-myc tag, Herpes simplex virusglycoprotein D (gD) tag, FLAG tag, KT3 epitope tag, tubulin epitope tag,T7 gene 10 protein peptide tag, streptavidin tag, streptavidin bindingpeptide (SPB) tag, Strep-tag, Strep-tag II, albumin-binding protein(ABP) tag, alkaline phosphatase (AP) tag, bluetongue virus tag (B-tag),calmodulin binding peptide (CBP) tag, chloramphenicol acetyl transferase(CAT) tag, choline-binding domain (CBD) tag, chitin binding domain (CBD)tag, cellulose binding domain (CBP) tag, dihydrofolate reductase (DHFR)tag, galactose-binding protein (GBP) tag, maltose binding protein (MBP),glutathione-S-transferase (GST), Glu-Glu (EE) tag, human influenzahemagglutinin (HA) tag, horseradish peroxidase (HRP) tag, NE-tag, HSVtag, ketosteroid isomerase (KSI) tag, KT3 tag, LacZ tag, luciferase tag,NusA tag, PDZ domain tag, AviTag, Calmodulin-tag, E-tag, S-tag, SBP-tag,Softag 1, Softag 3, TC tag, VSV-tag, Xpress tag, Isopeptag, SpyTag,SnoopTag, Profinity eXact tag, Protein C tag, S1-tag, S-tag,biotin-carboxy carrier protein (BCCP) tag, green fluorescent protein(GFP) tag, small ubiquitin-like modifier (SUMO) tag, tandem affinitypurification (TAP) tag, HaloTag, Nus-tag, Thioredoxin-tag, Fc-tag, CYDtag, HPC tag, TrpE tag, ubiquitin tag, VSV-G epitope tag, V5 tag, and acombination thereof; optionally, wherein the first or second affinityacceptor peptide comprises two or more repeats of a tag sequence.

In some embodiments, the linker comprises a polynucleic acid sequenceencoding a cleavable linker. In some embodiments, the cleavable linkeris a ribosomal skipping site or an internal ribosomal entry site (IRES)element. In some embodiments, the ribosomal skipping site or IRES iscleaved when expressed in the cells. In some embodiments, the ribosomalskipping site is selected from the group consisting of F2A, T2A, P2A,and E2A. In some embodiments, the IRES element is selected from commoncellular or viral IRES sequences.

In some embodiments, the determining comprises performing biochemicalanalysis or mass spectrometry, such as tandem mass spectrometry. In someembodiments, the determining comprises obtaining a peptide sequence thatcorresponds to an MS/MS spectra of one or more peptides isolated fromthe enriched affinity acceptor tagged HLA-peptide complexes from apeptide database; wherein one or more sequences obtained identifies thesequence of the one or more peptides. In some embodiments, the peptidedatabase is a no-enzyme specificity peptide database, such as a withoutmodification database or a with modification database. In someembodiments, the method further comprises searching the peptide databaseusing a reversed-database search strategy. In some embodiments, thepopulation of cells is a cell line. In some embodiments, the populationof cells is a human cell line. In some embodiments, the population ofcells is a mouse cell line. In some embodiments, the population of cellsis a CHO cell line. In some embodiments, the population of cells is acell line selected from HEK293T, expi293, HeLa, A375, 721.221, JEG-3,K562, Jurkat, Hep G2, SH-SY5Y, CACO-2, U937, U-2 OS, ExpiCHO, CHO andTHP1.

In some embodiments, the population of cells is treated with one or morecytokines, checkpoint inhibitors, epigenetically-active drugs, IFN-γ,agents that alter antigen processing (such as peptidase inhibitors,proteasome inhibitors, and TAP inhibitors), or a combination thereof. Insome embodiments, the population of cells is treated with one or morereagents that modulate a metabolic pathway or a metabolic status of thecells. In some embodiments, the population of cells is treated with oneor more reagents that modulate the cellular proteome of the cells. Insome embodiments, the population of cells is treated with one or morereagents that modulate or regulate cellular expression or transcription(e.g. AIRE or a CREB binding protein or modulators thereof) of thecells. In some embodiments, the population of cells is treated with oneor more reagents that modulate or regulate a transcription factor of thecells. In some embodiments, the population of cells is treated with oneor more reagents that modulate or regulate cellular expression ortranscription of an HLA of the cells. In some embodiments, thepopulation of cells is treated with one or more reagents that modulateor regulate cellular expression or transcription of the proteome of thecells.

In some embodiments, the population of cells comprises at least 10⁵cells, at least 10⁶ cells or at least 10⁷ cells. In some embodiments,the population of cells is a population of dendritic cells, macrophages,cancer cells or B-cells. In some embodiments, the population of cellscomprises tumor cells. In some embodiments, the population of cells iscontacted with an agent prior to isolating said HLA-peptide complexesfrom the one or more cells. In some embodiments, said agent is aninflammatory cytokine, a chemical agent, an adjuvant, a therapeuticagent or radiation.

In some embodiments, the HLA allele is a mutated HLA allele. In someembodiments, the sequence encoding the HLA allele comprises a barcodesequence. In some embodiments, the method further comprises assaying forexpression of the affinity acceptor tagged class I or class II HLAallele. In some embodiments, the assaying comprises assaying comprisessequencing an affinity acceptor tagged class I or class II HLA allele,detecting affinity acceptor tagged class I or class II HLA allele RNA,detecting affinity acceptor tagged class I or class II HLA alleleprotein, or a combination thereof. In some embodiments, assaying forexpression can comprise a Western blot assay, fluorescent activated cellsorting (FACS), mass spectrometry (MS), a microarray hybridizationassay, an RNA-seq assay, a polymerase chain reaction assay, a LAMPassay, a ligase chain reaction assay, a Southern blot assay, a Northernblot assay, or an enzyme-linked immunosorbent assay (ELISA).

In some embodiments, the method comprises carrying out the steps of themethod for different HLA alleles. In some embodiments, each differentHLA allele comprises a unique barcode sequence. In some embodiments,each polynucleic acid encoding a different HLA allele comprises a uniquebarcode sequence.

Provided herein is a HLA-allele specific binding peptide sequencedatabase obtained by carrying out a method described herein. Providedherein is a combination of two or more HLA-allele specific bindingpeptide sequence databases obtained by carrying out a method describedherein repeatedly, each time using a different HLA-allele. Providedherein is a method for generating a prediction algorithm for identifyingHLA-allele specific binding peptides, comprising training a machine witha peptide sequence database described herein or a combination describedherein.

In some embodiments, the machine combines one or more linear models,support vector machines, decision trees and neural networks. In someembodiments, a variable used to train the machine comprises one or morevariables selected from the group consisting of peptide sequence, aminoacid physical properties, peptide physical properties, expression levelof the source protein of a peptide within a cell, protein stability,protein translation rate, ubiquitination sites, protein degradationrate, translational efficiencies from ribosomal profiling, proteincleavability, protein localization, motifs of host protein thatfacilitate TAP transport, host protein is subject to autophagy, motifsthat favor ribosomal stalling, and protein features that favor NMD.

In some embodiments, the motifs that favor ribosomal stalling comprisepolyproline or polylysine stretches. In some embodiments, the proteinfeatures that favor NMD are selected from the group consisting of a long3′ UTR, a stop codon greater than 50 nucleic acids upstream of lastexon:exon junction, and peptide cleavability.

Provided herein is a method for identifying HLA-allele specific bindingpeptides comprising analyzing the sequence of a peptide with a machinewhich has been trained with a peptide sequence database obtained bycarrying out a method described herein for the HLA-allele. In someembodiments, the method comprises determining the expression level ofthe source protein of the peptide within a cell; and wherein the sourceprotein expression is a predictive variable used by the machine. In someembodiments, the expression level is determined by measuring the amountof source protein or the amount of RNA encoding said source protein.

Provided herein is a composition comprising a recombinant polynucleicacid comprising two or more sequences each encoding an affinity acceptortagged HLA, wherein the sequences encoding the affinity acceptor taggedHLAs comprise a sequence encoding a different recombinant HLA class Iα-chain allele, a sequence encoding an affinity acceptor peptide, andoptionally, a sequence encoding β2 microglobulin; wherein the sequencesof (a) and (b), and optionally (c), are operatively linked.

Provided herein is a composition comprising a recombinant polynucleicacid comprising two or more sequences each comprising a sequenceencoding an affinity acceptor tagged HLA, wherein the sequences encodingthe affinity acceptor tagged HLAs comprise a sequence encoding arecombinant HLA class II α-chain allele, a sequence encoding an affinityacceptor peptide, and optionally, a sequence encoding a HLA class IIβ-chain; wherein the sequences of (a) and (b), and optionally (c), areoperatively linked. In some embodiments, the recombinant polynucleicacid is isolated. In some embodiments, the class I HLA is selected fromthe group consisting of HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, and HLA-G. Insome embodiments, the class II HLA is selected from the group consistingof HLA-DR, HLA-DQ, and HLA-DP.

In some embodiments, the sequence encoding the affinity acceptor peptideis operatively linked to a sequence that encodes for an extracellularportion of the recombinant HLA allele. In some embodiments, the sequenceencoding the affinity acceptor molecule is operatively linked to theN-terminus of the sequence encoding the recombinant HLA allele. In someembodiments, the sequence encoding the affinity acceptor peptide isoperatively linked to a sequence encoding an intracellular portion ofthe recombinant HLA allele. In some embodiments, the sequence encodingthe affinity acceptor peptide is operatively linked to the C-terminus ofthe sequence encoding the recombinant HLA allele. In some embodiments,the sequence encoding the affinity acceptor peptide is operativelylinked to the sequence encoding the recombinant HLA allele by a linker.

In some embodiments, the two or more sequences encoding an affinityacceptor tagged HLA are expressed from the same polynucleotide. In someembodiments, the two or more sequences encoding an affinity acceptortagged HLA are expressed from different polynucleotides. In someembodiments, the encoded affinity acceptor peptide binds specifically toan affinity acceptor peptide binding molecule. In some embodiments, thetwo or more sequences encoding an affinity acceptor tagged HLA comprisetwo or more affinity acceptor peptides. In some embodiments, the two ormore sequences encoding an affinity acceptor tagged HLA comprise threeor more sequences encoding an affinity acceptor tagged HLA, wherein atleast two of the three or more sequences encoding an affinity acceptortagged HLA comprises the same affinity acceptor peptide. In someembodiments, the two or more affinity acceptor peptides are unique foreach of the two or more sequences encoding an affinity acceptor taggedHLA.

In some embodiments, the encoded affinity acceptor peptide is selectedfrom the group consisting of biotin acceptor peptide (BAP),poly-histidine tag, poly-histidine-glycine tag, poly-arginine tag,poly-aspartate tag, poly-cysteine tag, poly-phenylalanine, c-myc tag,Herpes simplex virus glycoprotein D (gD) tag, FLAG tag, KT3 epitope tag,tubulin epitope tag, T7 gene 10 protein peptide tag, streptavidin tag,streptavidin binding peptide (SPB) tag, Strep-tag, Strep-tag II,albumin-binding protein (ABP) tag, alkaline phosphatase (AP) tag,bluetongue virus tag (B-tag), calmodulin binding peptide (CBP) tag,chloramphenicol acetyl transferase (CAT) tag, choline-binding domain(CBD) tag, chitin binding domain (CBD) tag, cellulose binding domain(CBP) tag, dihydrofolate reductase (DHFR) tag, galactose-binding protein(GBP) tag, maltose binding protein (MBP), glutathione-S-transferase(GST), Glu-Glu (EE) tag, human influenza hemagglutinin (HA) tag,horseradish peroxidase (HRP) tag, NE-tag, HSV tag, ketosteroid isomerase(KSI) tag, KT3 tag, LacZ tag, luciferase tag, NusA tag, PDZ domain tag,AviTag, Calmodulin-tag, E-tag, S-tag, SBP-tag, Softag 1, Softag 3, TCtag, VSV-tag, Xpress tag, Isopeptag, SpyTag, SnoopTag, Profinity eXacttag, Protein C tag, S1-tag, S-tag, biotin-carboxy carrier protein (BCCP)tag, green fluorescent protein (GFP) tag, small ubiquitin-like modifier(SUMO) tag, tandem affinity purification (TAP) tag, HaloTag, Nus-tag,Thioredoxin-tag, Fc-tag, CYD tag, HPC tag, TrpE tag, ubiquitin tag,VSV-G epitope tag, V5 tag, and a combination thereof; optionally,wherein the first or second affinity acceptor peptide comprises two ormore repeats of a tag sequence.

In some embodiments, the affinity acceptor peptide binding molecule isbiotin or an antibody specific to the affinity acceptor peptide. In someembodiments, the affinity acceptor peptide binding molecule bindsspecifically to an affinity molecule. In some embodiments, the affinitymolecule is streptavidin, NeutrAvidin, or a derivative thereof. In someembodiments, the affinity acceptor peptide binding molecule does notspecifically interact with an amino acid sequence of the recombinantclass I or class II HLA. In some embodiments, for two or more of therecombinant polynucleic acids: the sequence encoding the affinityacceptor tagged HLA is stably integrated into the genome of a cell. Insome embodiments, the sequence encoding β2 microglobulin or the sequenceencoding the HLA class II β-chain is connected to a sequence encoding asecond affinity acceptor peptide. In some embodiments, the secondaffinity acceptor peptide comprises an HA tag. In some embodiments, thesequence encoding β2 microglobulin or the sequence encoding the HLAclass II β-chain is connected to the sequence encoding the recombinantHLA and the affinity acceptor peptide by a linker.

In some embodiments, the linker comprises a polynucleic acid sequenceencoding a cleavable linker. In some embodiments, the cleavable linkeris a ribosomal skipping site or an internal ribosomal entry site (IRES)element. In some embodiments, the ribosomal skipping site or IRES iscleaved when expressed in the cells. In some embodiments, the ribosomalskipping site is selected from the group consisting of F2A, T2A, P2A,and E2A In some embodiments, the IRES element is selected from commoncellular or viral IRES sequences.

Provided herein is a composition comprising two or more isolatedpolypeptide molecules encoded by the polynucleic acid of a compositiondescribed herein. Provided herein is a composition comprising apopulation of cells comprising two or more polypeptide molecules encodedby the polynucleic acid of a composition described herein. Providedherein is a composition comprising a population of cells comprising acomposition described herein. Provided herein is a compositioncomprising a population of cells comprising one or more cells comprisinga composition described herein.

In some embodiments, the population of cells express one or moreendogenous class I or class II HLA alleles. In some embodiments, thepopulation of cells are engineered to lack one or more endogenous HLAclass I alleles. In some embodiments, the population of cells areengineered to lack endogenous HLA class I alleles. In some embodiments,the population of cells are engineered to lack one or more endogenousHLA class II alleles. In some embodiments, the population of cells areengineered to lack endogenous HLA class II alleles. In some embodiments,the population of cells are engineered to lack one or more endogenousHLA class I alleles and one or more endogenous HLA class II alleles. Insome embodiments, the population of cells is a population of low cellsurface HLA class I or class II expressing cells. In some embodiments,the composition is formulated using peptides or polynucleic acidsencoding peptides specific to an HLA type of a patient. Provided hereinis a method of making a cell comprising transducing or transfecting twoor more cells with the two or more polynucleic acids of a compositiondescribed herein.

Provided herein is a peptide identified according to a method describedherein. Provided herein is a method of inducing an anti-tumor responsein a mammal comprising administering to the mammal an effective amountof a polynucleic acid comprising a sequence of a peptide describedherein. Provided herein is a method of inducing an anti-tumor responsein a mammal comprising administering to the mammal an effective amountof a peptide comprising the sequence of a peptide described herein.Provided herein is a method of inducing an anti-tumor response in amammal comprising administering to the mammal a cell comprising apeptide comprising the sequence of a peptide described herein. Providedherein is a method of inducing an anti-tumor response in a mammalcomprising administering to the mammal a cell comprising an effectiveamount of a polynucleic acid comprising a sequence encoding a peptidecomprising the sequence of a peptide described herein. In someembodiments, the cell presents the peptide as an HLA-peptide complex.Provided herein is a method of for inducing an immune response in amammal comprising administering to the mammal an effective amount of apolynucleic acid comprising a sequence encoding a peptide describedherein. Provided herein is a method for inducing an immune response in amammal comprising administering to the mammal an effective amount of apeptide comprising the sequence of a peptide described herein. Providedherein is a method for inducing an immune response in a mammalcomprising administering to the mammal an effective amount of a cellcomprising a peptide comprising the sequence of a peptide describedherein. Provided herein is a method for inducing an immune response in amammal comprising administering to the mammal an effective amount of acell comprising a polynucleic acid comprising a sequence encoding apeptide comprising the sequence of a peptide described herein.

In some embodiments, the immune response is a T cell immune response. Insome embodiments, the immune response is a CD8 T cell response. In someembodiments, the immune response is a CD4 T cell response. In someembodiments, the immune response is humoral immune response.

Provided herein is a method for treating a mammal having a diseasecomprising administering to the mammal an effective amount of apolynucleic acid comprising a sequence encoding a peptide describedherein. Provided herein is a method for treating a mammal having adisease comprising administering to the mammal an effective amount of apeptide comprising the sequence of a peptide described herein. Providedherein is a method for treating a mammal having a disease comprisingadministering to the mammal an effective amount of a cell comprising apeptide comprising the sequence of a peptide described herein. Providedherein is a method for treating a mammal having a disease comprisingadministering to the mammal an effective amount of a cell comprising apolynucleic acid comprising a sequence encoding a peptide comprising thesequence of a peptide described herein. In some embodiments, the diseaseis cancer. In some embodiments, the disease is infection by aninfectious agent. In some embodiments, the infectious agent is apathogen, optionally a virus or bacteria, or a parasite.

In some embodiments, the virus is selected from the group consisting of:BK virus (BKV), Dengue viruses (DENV-1, DENV-2, DENV-3, DENV-4, DENV-5),cytomegalovirus (CMV), Hepatitis B virus (HBV), Hepatitis C virus (HCV),Epstein-Barr virus (EBV), an adenovirus, human immunodeficiency virus(HIV), human T-cell lymphotrophic virus (HTLV-1), an influenza virus,RSV, HPV, rabies, mumps rubella virus, poliovirus, yellow fever,hepatitis A, hepatitis B, Rotavirus, varicella virus, humanpapillomavirus (HPV), smallpox, zoster, and any combination thereof.

In some embodiments, the bacteria is selected from the group consistingof: Klebsiella spp., Tropheryma whipplei, Mycobacterium leprae,Mycobacterium lepromatosis, and Mycobacterium tuberculosis, typhoid,pneumococcal, meningococcal, haemophilus B, anthrax, tetanus toxoid,meningococcal group B, bcg, cholera, and any combination thereof.

In some embodiments, the parasite is a helminth or a protozoan. In someembodiments, the parasite is selected from the group consisting of:Leishmania spp., Plasmodium spp., Trypanosoma cruzi, Ascarislumbricoides, Trichuris trichiura, Necator americanus, Schistosoma spp.,and any combination thereof.

Provided herein is a method of enriching for immunogenic peptidescomprising: providing a population of cells comprising one or more cellsexpressing an affinity acceptor tagged HLA, wherein the affinityacceptor tagged HLA comprises an affinity acceptor peptide operativelylinked to a recombinant HLA encoded by a recombinant HLA allele; andenriching for HLA-peptide complexes comprising the affinity acceptortagged HLA. In some embodiments, the method further comprisesdetermining the sequence of immunogenic peptides isolated from theHLA-peptide complexes. In some embodiments, the determining comprisesusing LC-MS/MS.

Provided herein is a method of treating a disease or disorder in asubject, the method comprising administering to the subject an effectiveamount of a polynucleic acid comprising a sequence encoding a peptidedescribed herein. Provided herein is a method of treating a disease ordisorder in a subject, the method comprising administering to thesubject an effective amount of a peptide comprising the sequence of apeptide described herein. Provided herein is a method of treating adisease or disorder in a subject, the method comprising administering tothe subject an effective amount of a cell comprising a peptidecomprising the sequence of a peptide described herein. Provided hereinis a method of treating a disease or disorder in a subject, the methodcomprising administering to the subject a cell comprising an effectiveamount of a polynucleic acid comprising a sequence encoding a peptidecomprising the sequence of a peptide described herein.

Provided herein is a method of developing an therapeutic for a subjectwith a disease or condition comprising providing a population of cellsderived from a subject with a disease or condition, expressing in one ormore cells of the population of cells an affinity acceptor tagged classI or class II HLA allele by introducing into the one or more cells apolynucleic acid encoding a sequence comprising: a sequence encoding arecombinant class I or class II HLA allele operatively linked to asequence encoding an affinity acceptor peptide, thereby forming affinityacceptor tagged HLA-peptide complexes in the one or more cells;enriching and characterizing the affinity acceptor tagged HLA-peptidecomplexes; and, optionally, developing an therapeutic based on thecharacterization.

Provided herein is a method of identifying at least one subject specificimmunogenic antigen and preparing a subject-specific immunogeniccomposition that includes the at least one subject specific immunogenicantigen, wherein the subject has a disease and the at least one subjectspecific immunogenic antigen is specific to the subject and thesubject's disease, said method comprising: providing a population ofcells derived from a subject with a disease or condition, expressing inone or more cells of the population of cells from the subject, anaffinity acceptor tagged class I or class II HLA allele by introducinginto the one or more cells a polynucleic acid encoding a sequencecomprising: a sequence encoding a recombinant class I or class II HLAallele operatively linked to a sequence encoding an affinity acceptorpeptide, thereby forming affinity acceptor tagged HLA-peptide complexesin the one or more cells; enriching affinity acceptor tagged HLA-peptidecomplexes from the one or more cells; identifying an immunogenic peptidefrom the enriched affinity acceptor tagged HLA-peptide complexes that isspecific to the subject and the subject's disease; and formulating asubject-specific immunogenic composition based one or more of thesubject specific immunogenic peptides identified.

In some embodiments, the therapeutic or subject specific immunogeniccomposition comprises a peptide from the enriched affinity acceptortagged HLA-peptide complexes or a or a polynucleotide encoding thepolypeptide from the enriched affinity acceptor tagged HLA-peptidecomplexes. In some embodiments, the therapeutic or subject specificimmunogenic composition comprises a T cell expressing a T cell receptor(TCR) that specifically binds to the polypeptide from the enrichedaffinity acceptor tagged HLA-peptide complexes. In some embodiments, thesubject specific immunogenic composition comprises a chimeric antigenreceptor (CAR) T cell expressing a receptor that specifically binds tothe polypeptide from the enriched affinity acceptor tagged HLA-peptidecomplexes.

In some embodiments, the method further comprises administering anothertherapeutic agent, optionally, an immune checkpoint inhibitor to thesubject. In some embodiments, the method further comprises administeringan adjuvant, optionally, poly-ICLC to the subject.

In some embodiments, the disease or disorder is cancer. In someembodiments, the disease or disorder is an autoimmune disease. In someembodiments, the disease or disorder is an infection. In someembodiments, the infection is an infection by an infectious agent. Insome embodiments, the infectious agent is a pathogen, a virus, bacteria,or a parasite.

In some embodiments, the virus is selected from the group consisting of:BK virus (BKV), Dengue viruses (DENV-1, DENV-2, DENV-3, DENV-4, DENV-5),cytomegalovirus (CMV), Hepatitis B virus (HBV), Hepatitis C virus (HCV),Epstein-Barr virus (EBV), an adenovirus, human immunodeficiency virus(HIV), human T-cell lymphotrophic virus (HTLV-1), an influenza virus,RSV, HPV, rabies, mumps rubella virus, poliovirus, yellow fever,hepatitis A, hepatitis B, Rotavirus, varicella virus, humanpapillomavirus (HPV), smallpox, zoster, and any combination thereof.

In some embodiments, the bacteria is selected from the group consistingof: Klebsiella spp., Tropheryma whipplei, Mycobacterium leprae,Mycobacterium lepromatosis, and Mycobacterium tuberculosis, typhoid,pneumococcal, meningococcal, haemophilus B, anthrax, tetanus toxoid,meningococcal group B, bcg, cholera, and combinations thereof.

In some embodiments, the parasite is a helminth or a protozoan. In someembodiments, the parasite is selected from the group consisting of:Leishmania spp., Plasmodium spp., Trypanosoma cruzi, Ascarislumbricoides, Trichuris trichiura, Necator americanus, Schistosoma spp.,and any combination thereof.

Provided herein is a method of developing a therapeutic for a subjectwith a disease or condition comprising: providing a population of cells,wherein one or more cells of the population of cells comprise apolynucleic acid comprising a sequence encoding at least two affinityacceptor tagged class I or class II HLA alleles, wherein the sequenceencoding the at least two affinity acceptor tagged class I or class IIHLAs comprises a first recombinant sequence comprising a sequenceencoding a first class I or class II HLA allele operatively linked to asequence encoding a first affinity acceptor peptide; and a secondrecombinant sequence comprising a sequence encoding a second class I orclass II HLA allele operatively linked to a sequence encoding a secondaffinity acceptor peptide; expressing the at least two affinity acceptortagged HLAs in at least one cell of the one or more cells of thepopulation of cells, thereby forming affinity acceptor taggedHLA-peptide complexes in the at least one cell; enriching for theaffinity acceptor tagged HLA-peptide complexes; and identifying apeptide from the enriched affinity acceptor tagged HLA-peptidecomplexes; and formulating an immunogenic composition based one or moreof the peptides identified, wherein the first and the second recombinantclass I or class II HLA alleles are matched to an HLA haplotype of asubject. In some embodiments, the subject has a disease or condition.

In some embodiments, the first recombinant class I or class II HLAallele is different than the second recombinant class I or class II HLAallele. In some embodiments, the first affinity acceptor peptide is thesame as the second affinity acceptor peptide. In some embodiments, themethod comprises characterizing a peptide bound to the first and/orsecond affinity acceptor tagged HLA-peptide complexes from theenriching. In some embodiments, the at least two affinity acceptortagged class I or class II HLA alleles comprise at least 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or50 class I and/or class II HLA alleles. In some embodiments, the firstand/or the second affinity acceptor tagged HLA-peptide complexescomprise a transmembrane domain. In some embodiments, the first and/orthe second affinity acceptor tagged HLA-peptide complexes comprise anintracellular domain. In some embodiments, the first and/or the secondaffinity acceptor tagged HLA-peptide complexes are not excreted. In someembodiments, the first and/or the second affinity acceptor taggedHLA-peptide complexes incorporate into a cell membrane when expressed.In some embodiments, the first and/or the second affinity acceptortagged HLA-peptide complexes are not soluble affinity acceptor taggedHLA-peptide complexes.

In some embodiments, the method further comprises generating anHLA-allele specific peptide database. In some embodiments, the methodcomprises introducing one or more exogenous peptides to the populationof cells. In some embodiments, the introducing comprises contacting thepopulation of cells with the one or more exogenous peptides orexpressing the one or more exogenous peptides in the population ofcells. In some embodiments, the introducing comprises contacting thepopulation of cells with one or more nucleic acids encoding the one ormore exogenous peptides.

In some embodiments, the one or more nucleic acids encoding the one ormore peptides is DNA. In some embodiments, the one or more nucleic acidsencoding the one or more peptides is RNA, optionally wherein the RNA ismRNA.

In some embodiments, the enriching does not comprise use of a tetramerreagent. In some embodiments, the method comprises determining thesequence of a peptide or a portion thereof bound to the first and/or thesecond affinity acceptor tagged HLA-peptide complex from the enriching.In some embodiments, the determining comprises biochemical analysis,mass spectrometry analysis, MS analysis, MS/MS analysis, LC-MS/MSanalysis, or a combination thereof.

In some embodiments, the method comprises evaluating a binding affinityor stability of a peptide or a portion thereof bound to the first and/orthe second affinity acceptor tagged HLA-peptide complex from theenriching. In some embodiments, the method comprises determining whethera peptide or a portion thereof bound to the first and/or the secondaffinity acceptor tagged HLA-peptide complex from the enriching containsone or more mutations. In some embodiments, the method comprisesevaluating associations of peptides with HLA molecules in the firstand/or the second affinity acceptor tagged HLA-peptide complex.

In some embodiments, the method comprises expressing a library ofpeptides in the population of cells, thereby forming a library ofaffinity acceptor tagged HLA-peptide complexes. In some embodiments, themethod comprises contacting to the population of cells a library ofpeptides or a library of sequences encoding peptides, thereby forming alibrary of affinity acceptor tagged HLA-peptide complexes. In someembodiments, the library comprises a library of peptides associated witha disease or condition.

In some embodiments, the disease or condition is cancer or an infectionwith an infectious agent. In some embodiments, the method comprisesintroducing the infectious agent or portions thereof into one or morecells of the population of cells. In some embodiments, the methodcomprises characterizing one or more peptides from the first and/or thesecond HLA-peptide complexes, optionally wherein the peptides are fromone or more target proteins of the infectious agent. In someembodiments, the method comprises characterizing one or more regions ofthe peptides from the one or more target proteins of the infectiousagent. In some embodiments, the method comprises identifying peptidesfrom the first and/or the second HLA-peptide complexes derived from aninfectious agent.

In some embodiments, the population of cells is from a biological samplefrom a subject with a disease or condition. In some embodiments, thepopulation of cells is a cell line. In some embodiments, the populationof cells is a population of primary cells. In some embodiments, thepeptide from the first and/or the second affinity acceptor taggedHLA-peptide complex is capable of activating a T cell from a subjectwhen presented by an antigen presenting cell. In some embodiments, themethod comprises comparing HLA-peptide complexes from diseased cells toHLA-peptide complexes from non-diseased cells. In some embodiments, themethod further comprises isolating peptides from the first and/or thesecond affinity acceptor tagged HLA-peptide complexes before theidentifying. In some embodiments, the population of cells is apopulation of low cell surface HLA class I or class II expressing cells.

In some embodiments, the population of cells expresses one or moreendogenous HLA alleles. In some embodiments, the population of cellsexpresses the endogenous HLA alleles normally expressed by thepopulation of cells. In some embodiments, the population of cells is anengineered population of cells lacking one or more endogenous HLA classI alleles. In some embodiments, the population of cells is an engineeredpopulation of cells lacking endogenous HLA class I alleles. In someembodiments, the population of cells is an engineered population ofcells lacking one or more endogenous HLA class II alleles. In someembodiments, the population of cells is an engineered population ofcells lacking endogenous HLA class II alleles. In some embodiments, thepopulation of cells is an engineered population of cells lackingendogenous HLA class I alleles and endogenous HLA class II alleles. Insome embodiments, the population of cells is a knock-out of one or moreHLA class I alleles. In some embodiments, the population of cells is aknock-out of one or more HLA class II alleles. In some embodiments, thepopulation of cells is a knock-out of all HLA class I alleles. In someembodiments, the population of cells is a knock-out of all HLA class IIalleles. In some embodiments, the population of cells is a knock-out ofall HLA class I alleles and a knock-out of all HLA class II alleles. Insome embodiments, the sequence encoding the at least two affinityacceptor tagged class I or class II HLA alleles encodes a class I HLA.In some embodiments, the class I HLA is selected from the groupconsisting of HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, and HLA-G. In someembodiments, the first recombinant class I or class II HLA allele is afirst class I HLA allele and the second recombinant class I or class IIHLA allele is a second class I HLA allele. In some embodiments, thesequence encoding the at least two affinity acceptor tagged class I orclass II HLA alleles encodes a class II HLA. In some embodiments, theclass II HLA is selected from the group consisting of HLA-DR, HLA-DQ,and HLA-DP. In some embodiments, the class II HLA comprises a HLA classII α-chain, a HLA class II β-chain, or a combination thereof. In someembodiments, the first recombinant class I or class II HLA allele is afirst class II HLA allele and the second recombinant class I or class IIHLA allele is a second class II HLA allele.

In some embodiments, the first sequence and the second sequence are eachoperatively linked. In some embodiments, the first sequence and thesecond sequence are comprised on different polynucleotide molecules. Insome embodiments, the sequence encoding the first and/or second affinityacceptor peptide is operatively linked to a sequence that encodes anextracellular portion of the first and/or second class I or class II HLAallele. In some embodiments, the first and/or second encoded affinityacceptor peptide is expressed extracellularly. In some embodiments, thesequence encoding the first and/or second affinity acceptor peptide isoperatively linked to the N-terminus of the sequence encoding the firstand/or second class I or class II HLA allele. In some embodiments, thesequence encoding the first and/or second affinity acceptor peptide isoperatively linked to a sequence that encodes an intracellular portionof the first and/or second class I or class II HLA allele. In someembodiments, the encoded first and/or second affinity acceptor peptideis expressed intracellularly. In some embodiments, the sequence encodingthe first and/or second affinity acceptor peptide is operatively linkedto the C-terminus of the sequence encoding the first and/or second classI or class II HLA allele. In some embodiments, the sequence encoding thefirst and/or second affinity acceptor peptide is operatively linked tothe sequence encoding the first and/or second class I or class II HLAallele by a linker.

In some embodiments, enriching comprises enriching for intact cellsexpressing the first and/or second affinity acceptor tagged HLA-peptidecomplexes. In some embodiments, the method does not comprise lysing thecells before enriching. In some embodiments, the method furthercomprises lysing the one or more cells before enriching. In someembodiments, enriching comprises contacting an affinity acceptor peptidebinding molecule to the first and/or second affinity acceptor taggedHLA-peptide complexes, wherein the affinity acceptor peptide bindingmolecule binds specifically to the first and/or second affinity acceptorpeptide.

In some embodiments, the first and/or second affinity acceptor peptidecomprises a tag sequence comprising a biotin acceptor peptide (BAP),poly-histidine tag, poly-histidine-glycine tag, poly-arginine tag,poly-aspartate tag, poly-cysteine tag, poly-phenylalanine, c-myc tag,Herpes simplex virus glycoprotein D (gD) tag, FLAG tag, KT3 epitope tag,tubulin epitope tag, T7 gene 10 protein peptide tag, streptavidin tag,streptavidin binding peptide (SPB) tag, Strep-tag, Strep-tag II,albumin-binding protein (ABP) tag, alkaline phosphatase (AP) tag,bluetongue virus tag (B-tag), calmodulin binding peptide (CBP) tag,chloramphenicol acetyl transferase (CAT) tag, choline-binding domain(CBD) tag, chitin binding domain (CBD) tag, cellulose binding domain(CBP) tag, dihydrofolate reductase (DHFR) tag, galactose-binding protein(GBP) tag, maltose binding protein (MBP), glutathione-S-transferase(GST), Glu-Glu (EE) tag, human influenza hemagglutinin (HA) tag,horseradish peroxidase (HRP) tag, NE-tag, HSV tag, ketosteroid isomerase(KSI) tag, KT3 tag, LacZ tag, luciferase tag, NusA tag, PDZ domain tag,AviTag, Calmodulin-tag, E-tag, S-tag, SBP-tag, Softag 1, Softag 3, TCtag, VSV-tag, Xpress tag, Isopeptag, SpyTag, SnoopTag, Profinity eXacttag, Protein C tag, S1-tag, S-tag, biotin-carboxy carrier protein (BCCP)tag, green fluorescent protein (GFP) tag, small ubiquitin-like modifier(SUMO) tag, tandem affinity purification (TAP) tag, HaloTag, Nus-tag,Thioredoxin-tag, Fc-tag, CYD tag, HPC tag, TrpE tag, ubiquitin tag,VSV-G epitope tag, V5 tag, or a combination thereof optionally, whereinthe first and/or second affinity acceptor peptide comprises two or morerepeats of a tag sequence.

In some embodiments, the affinity acceptor peptide binding molecule isbiotin or an antibody specific to the first and/or second affinityacceptor peptide. In some embodiments, the enriching comprisescontacting an affinity molecule to the first and/or second affinityacceptor tagged HLA-peptide complexes, wherein the affinity moleculebinds specifically to the affinity acceptor peptide binding molecule. Insome embodiments, the affinity molecule is streptavidin, NeutrAvidin, ora derivative thereof. In some embodiments, enriching comprisesimmunoprecipitating the first and/or second affinity acceptor taggedHLA-peptide complexes.

In some embodiments, the affinity acceptor peptide binding molecule isattached to a solid surface. In some embodiments, the affinity moleculeis attached to a solid surface. In some embodiments, the solid surfaceis a bead.

In some embodiments, enriching comprises immunoprecipitating the firstand/or second affinity acceptor tagged HLA-peptide complexes with anaffinity acceptor peptide binding molecule that binds specifically tothe first and/or second affinity acceptor peptide. In some embodiments,the affinity acceptor peptide binding molecule does not specificallyinteract with the amino acid sequence of the encoded first and/or secondclass I or class II HLA. In some embodiments, enriching comprisescontacting an affinity molecule specific to an extracellular portion ofthe first and/or second class I or class II HLA allele. In someembodiments, enriching comprises contacting an affinity moleculespecific to an N-terminal portion of the first and/or second class I orclass II HLA allele.

In some embodiments, providing comprises contacting the population ofcells with the polynucleic acid. In some embodiments, contactingcomprises transfecting or transducing. In some embodiments, providingcomprises contacting the population of cells with a vector comprisingthe polynucleic acid. In some embodiments, the vector is a viral vector.In some embodiments, the polynucleic acid is stably integrated into thegenome of the population of cells.

In some embodiments, the sequence encoding the first and/or second classI or class II HLA comprises a sequence encoding a HLA class I α-chain.In some embodiments, the first recombinant class I or class II HLAallele is a first HLA class I α-chain and the second recombinant class Ior class II HLA allele is a second HLA class I α-chain.

In some embodiments, the method further comprises expressing a sequenceencoding β2 microglobulin in the one or more cells. In some embodiments,the sequence encoding β2 microglobulin is connected to the sequenceencoding the first and/or second class I or class II HLA. In someembodiments, the sequence encoding β2 microglobulin is connected to thesequence encoding the first and/or second class I or class II HLA by alinker. In some embodiments, the sequence encoding β2 microglobulin isconnected to a sequence encoding a third affinity acceptor peptide.

In some embodiments, the third affinity acceptor peptide is differentthan the first and/or second affinity acceptor peptide. In someembodiments, the sequence encoding the first and/or second class I orclass II HLA comprises a sequence encoding a HLA class II α-chain and/ora HLA class II β-chain. In some embodiments, the sequence encoding thefirst and/or second class I or class II HLA comprises a sequenceencoding a first HLA class II α-chain and a second HLA class II α-chain.In some embodiments, the method further comprises expressing a sequenceencoding a HLA class II β-chain in the one or more cells. In someembodiments, the sequence encoding a first HLA class II α-chain and asecond HLA class II α-chain HLA is connected to the sequence encodingthe HLA class II β-chain. In some embodiments, the sequence encoding thefirst and/or second class I or class II HLA comprises a sequenceencoding a first HLA class II β-chain and a second HLA class II β-chain.

In some embodiments, the method further comprises expressing a sequenceencoding a HLA class II α-chain in the one or more cells. In someembodiments, the sequence encoding a first HLA class II β-chain and asecond HLA class II β-chain is connected to the sequence encoding theHLA class II α-chain by a linker. In some embodiments, the sequenceencoding the HLA class II β-chain or the HLA class II α-chain isconnected to a sequence encoding a third affinity acceptor peptide. Insome embodiments, the third affinity acceptor peptide is different thanthe first and/or second affinity acceptor peptide.

In some embodiments, the third affinity acceptor peptide is differentthan the first affinity acceptor peptide and is selected from the groupconsisting of biotin acceptor peptide (BAP), poly-histidine tag,poly-histidine-glycine tag, poly-arginine tag, poly-aspartate tag,poly-cysteine tag, poly-phenylalanine, c-myc tag, Herpes simplex virusglycoprotein D (gD) tag, FLAG tag, KT3 epitope tag, tubulin epitope tag,T7 gene 10 protein peptide tag, streptavidin tag, streptavidin bindingpeptide (SPB) tag, Strep-tag, Strep-tag II, albumin-binding protein(ABP) tag, alkaline phosphatase (AP) tag, bluetongue virus tag (B-tag),calmodulin binding peptide (CBP) tag, chloramphenicol acetyl transferase(CAT) tag, choline-binding domain (CBD) tag, chitin binding domain (CBD)tag, cellulose binding domain (CBP) tag, dihydrofolate reductase (DHFR)tag, galactose-binding protein (GBP) tag, maltose binding protein (MBP),glutathione-S-transferase (GST), Glu-Glu (EE) tag, human influenzahemagglutinin (HA) tag, horseradish peroxidase (HRP) tag, NE-tag, HSVtag, ketosteroid isomerase (KSI) tag, KT3 tag, LacZ tag, luciferase tag,NusA tag, PDZ domain tag, AviTag, Calmodulin-tag, E-tag, S-tag, SBP-tag,Softag 1, Softag 3, TC tag, VSV-tag, Xpress tag, Isopeptag, SpyTag,SnoopTag, Profinity eXact tag, Protein C tag, S1-tag, S-tag,biotin-carboxy carrier protein (BCCP) tag, green fluorescent protein(GFP) tag, small ubiquitin-like modifier (SUMO) tag, tandem affinitypurification (TAP) tag, HaloTag, Nus-tag, Thioredoxin-tag, Fc-tag, CYDtag, HPC tag, TrpE tag, ubiquitin tag, VSV-G epitope tag, V5 tag, and acombination thereof; optionally, wherein the first or second affinityacceptor peptide comprises two or more repeats of a tag sequence.

In some embodiments, the linker comprises a polynucleic acid sequenceencoding a cleavable linker. In some embodiments, the cleavable linkeris a ribosomal skipping site or an internal ribosomal entry site (IRES)element. In some embodiments, the ribosomal skipping site or IRES iscleaved when expressed in the cells. In some embodiments, the ribosomalskipping site is selected from the group consisting of F2A, T2A, P2A,and E2A. In some embodiments, the IRES element is selected from commoncellular or viral IRES sequences.

In some embodiments, the method comprises performing biochemicalanalysis or mass spectrometry, such as tandem mass spectrometry. In someembodiments, the method comprises obtaining a peptide sequence thatcorresponds to an MS/MS spectra of one or more peptides isolated fromthe enriched affinity acceptor tagged HLA-peptide complexes from apeptide database; wherein one or more sequences obtained identifies thesequence of the one or more peptides.

In some embodiments, the population of cells is a cell line selectedfrom HEK293T, expi293, HeLa, A375, 721.221, JEG-3, K562, Jurkat, Hep G2,SH-SY5Y, CACO-2, U937, U-2 OS, ExpiCHO, CHO and THP1. In someembodiments, the cell line is treated with one or more cytokines,checkpoint inhibitors, epigenetically-active drugs, IFN-γ, or acombination thereof. In some embodiments, the population of cellscomprises at least 10⁵ cells, at least 10⁶ cells or at least 10⁷ cells.In some embodiments, the population of cells is a population ofdendritic cells, macrophages, cancer cells or B-cells. In someembodiments, the population of cells comprises tumor cells.

In some embodiments, the population of cells is contacted with an agentprior to isolating the first and/or second HLA-peptide complexes fromthe one or more cells. In some embodiments, the agent is an inflammatorycytokine, a chemical agent, an adjuvant, a therapeutic agent orradiation.

In some embodiments, the first and or second HLA allele is a mutated HLAallele. In some embodiments, the sequence encoding the first and orsecond HLA allele comprises a barcode sequence. In some embodiments, themethod further comprises assaying for expression of the first and/orsecond affinity acceptor tagged class I or class II HLA allele.

In some embodiments, the assaying comprises sequencing the first and/orsecond affinity acceptor tagged class I or class II HLA allele,detecting RNA encoding the first and/or second affinity acceptor taggedclass I or class II HLA allele RNA, detecting the first and/or secondaffinity acceptor tagged class I or class II HLA allele protein, or acombination thereof. In some embodiments, the first and second affinityacceptor tagged class I or class II HLA allele comprises a uniquebarcode sequence. In some embodiments, the first sequence and the secondsequence comprise a unique barcode sequence.

BRIEF DESCRIPTION OF THE DRAWINGS

The features of the present disclosure are set forth with particularityin the appended claims. A better understanding of the features andadvantages of the present disclosure will be obtained by reference tothe following detailed description that sets forth illustrativeembodiments, in which the principles of the disclosure are utilized, andthe accompanying drawings of which:

FIG. 1A is a representative schematic of a universal immunopurificationand data generation pipeline. Class I and/or class II HLA molecules areintroduced into any cell, including a cell not expressing class I orclass II HLA) so that specific class I or class II HLA allele(s) areexpressed in the cell. Populations of genetically engineered HLAexpressing cells are harvested, lysed, and their HLA-peptide complexesare tagged (e.g., biotinylated) and immunopurified (e.g., using thebiotin-streptavidin interaction). HLA-associated peptides specific to asingle HLA can be eluted from their tagged (e.g., biotinylated)complexes and evaluated (e.g., sequenced using high resolutionLC-MS/MS).

FIG. 1B is a schematic of structures of HLA class II molecules -DP, -DQ,and -DR. HLA-DR molecules are heterodimers containing a constant α-chainand a variable β-chain. HLA-DQ and HLA-DP molecules are heterodimerscontaining variable α-chains and variable β-chains.

FIG. 2 is a representative schematic of constructs designed for HLAclass I and II expression in cultured cell lines. HLA-A*02:01 constructsrepresent HLA class I design that implement biotin acceptor peptides(BAP) for biotinylation and immunopurification. HLA-DRB1*11:01constructs represent HLA class II design that implement biotin acceptorpeptides (BAP) for biotinylation and immunopurification.

FIG. 3 is a schematic of an exemplary lentiviral vector that can be usedto generate stable cell lines expressing HLA class I and class IIconstructs.

FIG. 4A is a representative schematic of a transfection-basedintroduction of class I or class II HLA constructs for universal IP andHLA-associated peptide sequencing by LC-MS/MS.

FIG. 4B is a representative schematic of a transfection-basedintroduction of class I or class II HLA constructs followed by aselection process, e.g., inclusion of an antibiotic resistance gene. Theselected cells can then be submitted for universal IP and HLA-associatedpeptide sequencing by LC-MS/MS.

FIG. 5 is a schematic of universal immunopurification for class I andclass II HLA. Cells, such as HEK293T (human embryonic kidney), areeither transfected or transduced to express a single class I or class IIHLA allele with an affinity tag for immunopurification. HLA-taggedexpressing cells are harvested, lysed, and their HLA-peptide complexesare biotinylated and immunopurified using the biotin-streptavidininteraction. HLA-associated peptides specific to a single HLA are elutedfrom their biotinylated complexes and analyzed (e.g., sequenced usinghigh resolution LC-MS/MS).

FIG. 6A is a Western blot (anti-biotinylation) comparing mock, GFP andempty plasmid transfections with HLA-A*02:01 constructs forbiotinylation-based immunoprecipitation demonstrating expression ofclass I HLA alleles in HEK293T cells.

FIG. 6B is a Ponceau stained gel used as a loading control for theWestern blot analysis.

FIG. 6C is a schematic representation of class I HLA constructs used togenerate engineered HEK293T cells imaged in FIG. 6A and FIG. 6B.

FIG. 7A is Western blot (top) and loading control (bottom) images of abiotinylation time course experiment demonstrating that C- andN-terminally labeled HLA-BAP biotinylation is complete in 10 minutes forboth class I and class II HLA-BAP expressing cells. The results showtransfection and biotinylation optimization of class I and class IIHLA-BAP alleles expressed by HEK293T cells.

FIG. 7B is a Western blot against the anti-BAP (top) and loading control(bottom) from cells expressing both N- and C-terminal BAP-labeled classI and class II HLA constructs.

FIG. 7C is a schematic representation of both N- and C-terminalBAP-labeled class I (HLA-A*02:01) and class II HLA-DRβ*11:01) constructsused for transfection and biotinylation optimization.

FIG. 8A is a Western blot image (anti-streptavidin for BAP label andanti-HA for HA label) and loading controls (Ponceau S) showing theexpression of biotinylated class I and class II HLA constructs used forHLA immunoprecipitation in HEK293T cells. Lysates were analyzed beforeaddition of biotin (−Biotin), after the addition of biotin (+BiotinInput), and after biotinylation and subsequent pulldown withstreptavidin beads (+Biotin FT). The reduction in signal in the +BiotinFT lane demonstrates that biotinylated MHC is being removed from thelysate and binding to the streptavidin beads.

FIG. 8B is a Western blot image (anti-streptavidin for BAP label andanti-HA for HA label) and loading controls (Ponceau S) showing theexpression of biotinylated class I and class II HLA constructs used forHLA immunoprecipitation in HeLa (human cervical cancer) cells. Lysateswere analyzed before addition of biotin (−Biotin), after the addition ofbiotin (+Biotin Input), and after biotinylation and subsequent pulldownwith streptavidin beads (+Biotin FT). The reduction in signal in the+Biotin FT lane demonstrates that biotinylated MHC is being removed fromthe lysate and binding to the streptavidin beads.

FIG. 8C is a Western blot image (anti-streptavidin for BAP label andanti-HA for HA label) and loading controls (Ponceau S) showing theexpression of biotinylated class I and class II HLA constructs used forHLA immunoprecipitation in A375 (human malignant melanoma) cells.Lysates were analyzed before addition of biotin (−Biotin), after theaddition of biotin (+Biotin Input), and after biotinylation andsubsequent pulldown with streptavidin beads (+Biotin FT). The reductionin signal in the +Biotin FT lane demonstrates that biotinylated MHC isbeing removed from the lysate and binding to the streptavidin beads.

FIG. 8D is a Western blot image (anti-streptavidin for BAP label andanti-HA for HA label) and loading controls (Ponceau S) showing theexpression of biotinylated class I and class II HLA constructs used forHLA immunoprecipitation in Expi293 cells (human embryonic kidneygenetically engineered for high density culture and protein expression).Lysates were analyzed before addition of biotin (−Biotin), after theaddition of biotin (+Biotin Input), and after biotinylation andsubsequent pulldown with streptavidin beads (+Biotin FT). The reductionin signal in the +Biotin FT lane demonstrates that biotinylated MHC isbeing removed from the lysate and binding to the streptavidin beads.

FIG. 9A is a bar graph of an exemplary LC-MS/MS analysis ofHLA-associated peptides isolated using the universal HLAimmunoprecipitation (Universal IP) pipeline. A bar plot representationof the total unique HLA-associated peptides identified from multiplecell types (A375; gray, HEK293T; orange, HeLa; blue) that expressaffinity-tagged class I and class II HLA constructs used in theUniversal IP pipeline is shown.

FIG. 9B is a bar plot showing representative data from class I HLAmono-allelic peptide profiling by LC-MS/MS. Each bar represents thetotal number of unique HLA-associated peptides identified from class Imono-allelic experiments that implemented the affinity-tagged HLAconstructs.

FIG. 9C is a bar plot showing representative data from class II HLAmono-allelic peptide profiling by LC-MS/MS. Each bar represents thetotal number of unique HLA-associated peptides identified from class IImono-allelic experiments that implemented the affinity-tagged HLAconstructs.

FIG. 10A is an exemplary schematic of the characteristics of class I andclass II HLA-associated peptides discovered using the Universal IPpipeline. An exemplary sequence logo representation of class IHLA-A*02:01-associated peptides and class II HLA-DRβ*11:01-associatedpeptides isolated and sequenced using the Universal IP platform isshown.

FIG. 10B is a bar graph showing HLA-associated peptide lengthdistributions comparing class I (red; HLA-A*02:01) and class II (blue;HLA-DRβ*11:01) HLA-associated peptides identified using the Universal IPpipeline. The length distributions of both class I and class IIHLA-associated peptides identified using the Universal IP follow theexpected trends.

FIG. 11A is a schematic representation of class II HLA constructs thatwere engineered for expression by different cell types for the UniversalIP pipeline.

FIG. 11B is a schematic representation of the class II HLA complexesthat can form upon expression of the construct shown in FIG. 11A in celllines expressing endogenous class II HLA α-chain and β-chain subunits.Class II HLA complexes are formed by α-chain and β-chain pairing, whichare each tagged with a different affinity handle.

FIG. 12A is a schematic representation of a serial Universal IP strategythat can be used for deconvolution of class II HLA α-chain and β-chainpairing depicted in FIG. 11B and unambiguous peptide-binding assignmentsto specific class II HLA complexes and demonstrates validation of serialuniversal IP of class II HLA complexes containing multiple affinitytags. Cells expressing dual-affinity tagged class II HLA constructs arelysed, biotinylated, and incubated with beads coupled to anti-HAantibodies. Class II HLA complexes with HA-tagged subunits are isolated,washed, and eluted using an HA peptide (e.g., YPYDVPDYA). The elution isthen incubated with beads coupled to either NeutrAvidin or streptavidinto isolate the HA-tagged and biotin-tagged class II HLA complexes.Peptides bound to dual-tagged class II HLA complexes are then eluted andsequenced by LC-MS/MS.

FIG. 12B is a Western blot validation of the serial Universal IPstrategy in HEK293T expressing dual-tagged HLA-DRB*11:01 constructs. Ananti-HA antibody was used to follow the serial enrichment process. Aloading control (Ponceau S stained gel) is shown.

FIG. 12C represents the results from an exemplary negative controlexperiment where cells expressing dual-affinity tagged class II HLAconstruct HLA-DRB*11:01 were lysed and incubated with beads coupled toanti-HA antibodies without biotinylation. A Western blot and loadingcontrol (Ponceau S stained gel) are shown to demonstrate the specificityof the serial Universal IP pipeline. No enrichment was observed when thebiotinylation step was removed from the serial Universal IP protocol.

FIG. 13 is a schematic representation of an overview HLA class IItrimming experiments that enable identification of core bindingepitopes. HLA class II molecules bind nested sets of peptides, usually12-18 amino acids in length, generated from the same source protein.Longer peptides overhang from the N- and C-terminal sides of HLA classII molecules, while the core epitope interacts most strongly withpeptide-binding groove. Peptides bound to HLA class II molecules aretrimmed using peptidases specific for N- and C-terminal ends. Aftertrimming, core peptide epitopes are sequenced using LC-MS/MS.

FIG. 14A is a schematic representation of a mono-allelic HLA-peptidomeprofiling approach that implements a biotin affinity tag. An exemplaryembodiment of the present disclosure makes use of the biotin acceptorpeptide (BAP) that is biotinylated on a lysine (K) residue by a BirAenzyme. The BAP peptide sequence contains a lysine residue that isbiotinylated upon the addition of BirA enzyme, biotin, and ATP. Thebiotinylated product displays high affinity forstreptavidin/NeutrAvidin. Streptavidin/NeutrAvidin beads can be used toenrich for the biotinylated BAP peptide sequence.

FIG. 14B is a schematic representation of biotin-basedimmunopurification of genetically engineered HLA molecules. A specificHLA allele with a BAP sequence at either the N- or C-terminus of the HLAprotein is introduced into a cell, e.g., by transfection or transductionof a plasmid. Note that the plasmid contains a DNA barcode that allowsfor a PCR-based method to monitor the cell line for each allele. Barcodelengths can be at least 5 base pairs, at least 10 base pairs, at least15 base pairs, at least 20 base pairs or more. Cells expressing theHLA-BAP proteins are lysed and biotinylated. HLA-BAP-peptide complexesare immunopurified from the complex lysate mixture, which can besubjected to LC-MS/MS analysis for peptide identification.

FIG. 15 is a schematic representation of an exemplary application of theUniversal IP platform for targeted epitope validation and discovery. Acell line of interest is engineered to express an allele-specificHLA-tagged (e.g., BAP) construct. Cells expressing HLA-tagged (e.g.,BAP) molecules are genetically engineered to express a single epitope ormultiple epitopes. Epitope expressing cells are lysed andHLA-BAP-peptide complexes are immunopurified. Isolated peptide antigenscan be examined by any suitable means, e.g., sequenced by LC-MS/MS, andpeptide fragments generated from the introduced epitopes can be used asa high-throughput readout for HLA-allele-matched antigen processing andpresentation.

FIG. 16 is a schematic representation of HLA allele multiplexing withinthe Universal IP pipeline. Multiple class I and class II alleles can beexpressed from a single HLA construct. For example, multiple heavychains can be included in a class I construct and multiple β- and/orα-chains can be included in a class II construct. By multiplexing HLAalleles in a single construct, multiple HLA molecules can be deliveredand expressed in a cell line of interest. Allele multiplexing enablesthe matching to patient HLA types and personalized peptide antigenreadouts with the application of the Universal IP pipeline andsubsequent complex and/or peptide analysis, e.g., LC-MS/MS readout.

FIG. 17 is schematics of multi-allelic and mono-allelic approaches inHLA ligand profiling. In a multi-allelic approach, the HLA ligands areco-immunoprecipitated with HLA heterodimers directly from patientmaterial or cell lines (top). Because these cells naturally expressedmultiple HLA alleles, peptides identified from such multi-allelicapproaches must be deconvoluted to assign binding to a specific HLAheterodimer if the HLA types are known. In a mono-allelic approach, theHLA-ligands are co-immunoprecipitated with HLA heterodimers from celllines genetically modified for expression of only a single HLA allele(bottom). Thus, peptides identified from mono-allelic approaches do notrequire deconvolution for HLA heterodimer binding assignments.

FIG. 18A is a diagram showing mutated neoantigenic peptide presented onMHC.

FIG. 18B is a schematic method of developing personalizedneoantigen-targeting therapy as described herein.

FIG. 19 shows a schematic showing different experimental approaches ofdifferent HLA-ligand profiling. Biochemical peptide:MHC (p:MHC) bindingassay is slow and low-throughput and has no insights on processing.Multi-allelic mass spectrometry is high-throughput and has ability tolearn processing rules; however, it requires in silico imputation toassign peptides to alleles. Mono-allelic mass spectrometry provides arapid, unbiased, and clean approach for defining peptide-binding motifsacross diverse WIC alleles. Mono-allelic mass spectrometry can rapidlyand systematically fill allege coverage gaps and makes it possible toleverage allele-specific peptide length preferences.

FIG. 20A shows a table of exemplary HLA binding peptides for A*01:01,B*51:01, A*29:02, and B*54:01 alleles uncovered using mono-allelicapproach. Mono-allelic approach uncovers HLA-binding peptides that arepoorly scored by NetMHCpan but biochemically validate as strong binders.

FIG. 20B is a bar graph showing rates of incorrect assignment in 100simulated deconvolutions. A random six allele patient HLA genotype (2alleles each of HLA-A, HLA-B, and HLA-C, sampling at US allelefrequencies) was generated. For each allele, 500 peptides from relevantmono-allelic experiment were sampled and combined to create mock 3000peptide multi-allelic data set. Each peptide was assigned to allele thatyields the best NetMHCpan % rank score to determine percentage ofpeptides incorrectly assigned by NetMHCpan. This process was repeated100 times.

FIG. 21 is a schematic illustration of MEW presentation predictor fordiverse individual MEW Class I alleles using MS data. Model training andevaluation are conducted on non-overlapping source proteins. MS-observedpeptides are assigned to train/test depending on source protein.Evaluation approach employs a 5000:1 excess of decoys to true binders.

FIG. 22 is a bar graph showing significantly improved predictions bothin terms of processing and allele-specific binding.

DETAILED DESCRIPTION

The following description and examples illustrate embodiments of thedisclosure in detail. It is to be understood that this disclosure is notlimited to the particular embodiments described herein and as such canvary. Those of skill in the art will recognize that there are numerousvariations and modifications of this disclosure, which are encompassedwithin its scope.

All terms are intended to be understood as they would be understood by aperson skilled in the art. Unless defined otherwise, all technical andscientific terms used herein have the same meaning as commonlyunderstood by one of ordinary skill in the art to which the disclosurepertains.

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described.

Although various features of the present disclosure can be described inthe context of a single embodiment, the features can also be providedseparately or in any suitable combination. Conversely, although thepresent disclosure can be described herein in the context of separateembodiments for clarity, the disclosure can also be implemented in asingle embodiment.

The following definitions supplement those in the art and are directedto the current application and are not to be imputed to any related orunrelated case, e.g., to any commonly owned patent or application.Although any methods and materials similar or equivalent to thosedescribed herein can be used in the practice for testing of the presentdisclosure, exemplary materials and methods are described herein.Accordingly, the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to belimiting.

Definitions

In this application, the use of the singular includes the plural unlessspecifically stated otherwise. It must be noted that, as used in thespecification, the singular forms “a,” “an” and “the” include pluralreferents unless the context clearly dictates otherwise. In thisapplication, the use of “or” means “and/or” unless stated otherwise.Furthermore, use of the term “including” as well as other forms, such as“include”, “includes,” and “included,” is not limiting.

The terms “one or more” or “at least one,” such as one or more or atleast one member(s) of a group of members, is clear per se, by means offurther exemplification, the term encompasses inter alia a reference toany one of said members, or to any two or more of said members, such as,e.g., any ≥3, ≥4, ≥5, ≥6 or ≥7 etc. of said members, and up to all saidmembers.

Reference in the specification to “some embodiments,” “an embodiment,”“one embodiment” or “other embodiments” means that a feature, structure,or characteristic described in connection with the embodiments isincluded in at least some embodiments, but not necessarily allembodiments, of the present disclosure.

As used in this specification and claim(s), the words “comprising” (andany form of comprising, such as “comprise” and “comprises”), “having”(and any form of having, such as “have” and “has”), “including” (and anyform of including, such as “includes” and “include”) or “containing”(and any form of containing, such as “contains” and “contain”) areinclusive or open-ended and do not exclude additional, unrecitedelements or method steps. It is contemplated that any embodimentdiscussed in this specification can be implemented with respect to anymethod or composition of the disclosure, and vice versa. Furthermore,compositions of the disclosure can be used to achieve methods of thedisclosure.

The term “about” or “approximately” as used herein when referring to ameasurable value such as a parameter, an amount, a temporal duration,and the like, is meant to encompass variations of +/−20% or less, +/−10%or less, +/−5% or less, or +/−1% or less of and from the specifiedvalue, insofar such variations are appropriate to perform in the presentdisclosure. It is to be understood that the value to which the modifier“about” or “approximately” refers is itself also specifically disclosed.

The term “immune response” includes T cell mediated and/or B cellmediated immune responses that are influenced by modulation of T cellcostimulation. Exemplary immune responses include T cell responses,e.g., cytokine production, and cellular cytotoxicity. In addition, theterm immune response includes immune responses that are indirectlyaffected by T cell activation, e.g., antibody production (humoralresponses) and activation of cytokine responsive cells, e.g.,macrophages.

A “receptor” is to be understood as meaning a biological molecule or amolecule grouping capable of binding a ligand. A receptor can serve totransmit information in a cell, a cell formation or an organism. Thereceptor comprises at least one receptor unit and can contain two ormore receptor units, where each receptor unit can consist of a proteinmolecule, e.g., a glycoprotein molecule. The receptor has a structurethat complements the structure of a ligand and can complex the ligand asa binding partner. Signaling information can be transmitted byconformational changes of the receptor following binding with the ligandon the surface of a cell. According to the present disclosure, areceptor can refer to proteins of MHC classes I and II capable offorming a receptor/ligand complex with a ligand, e.g., a peptide orpeptide fragment of suitable length.

A “barcode” sequence can be a nucleic acid sequence that can encode anitem of information about a sequence, such the identity of a sequence towhich the barcode is attached or the identity of a sample from which asequence is derived.

By “ligand” is meant a molecule which is capable of forming a complexwith a receptor. According to the present disclosure, a ligand is to beunderstood as meaning, for example, a peptide or peptide fragment whichhas a suitable length and suitable binding motives in its amino acidsequence, so that the peptide or peptide fragment is capable of forminga complex with proteins of MHC class I or MHC class II.

An “antigen” is a molecule capable of stimulating an immune response,and can be produced by cancer cells or infectious agents or anautoimmune disease. Antigens recognized by T cells, whether helper Tlymphocytes (T helper (T_(H)) cells) or cytotoxic T lymphocytes (CTLs),are not recognized as intact proteins, but rather as small peptides thatassociate with class I or class II MHC proteins on the surface of cells.During the course of a naturally occurring immune response, antigensthat are recognized in association with class II MHC molecules onantigen presenting cells (APCs) are acquired from outside the cell,internalized, and processed into small peptides that associate with theclass II MHC molecules. APCs can also cross-present peptide antigens byprocessing exogenous antigens and presenting the processed antigens onclass I MHC molecules. Antigens that give rise to proteins that arerecognized in association with class I MHC molecules are generallyproteins that are produced within the cells, and these antigens areprocessed and associate with class I MHC molecules. It is now understoodthat the peptides that associate with given class I or class II MHCmolecules are characterized as having a common binding motif, and thebinding motifs for a large number of different class I and II MHCmolecules have been determined. Synthetic peptides, that correspond tothe amino acid sequence of a given antigen and that contain a bindingmotif for a given class I or II MHC molecule, can also be synthesized.These peptides can then be added to appropriate APCs, and the APCs canbe used to stimulate a T helper cell or CTL response either in vitro orin vivo. The binding motifs, methods for synthesizing the peptides, andmethods for stimulating a T helper cell or CTL response are all knownand readily available to one of ordinary skill in the art.

The term “peptide” is used interchangeably with “mutant peptide” and“neoantigenic peptide” in the present specification. Similarly, the term“polypeptide” is used interchangeably with “mutant polypeptide” and“neoantigenic polypeptide” in the present specification. By “neoantigen”or “neoepitope” is meant a class of tumor antigens or tumor epitopeswhich arises from tumor-specific mutations in expressed protein. Thepresent disclosure further includes peptides that comprise tumorspecific mutations, peptides that comprise known tumor specificmutations, and mutant polypeptides or fragments thereof identified bythe method of the present disclosure. These peptides and polypeptidesare referred to herein as “neoantigenic peptides” or “neoantigenicpolypeptides.” The polypeptides or peptides can be a variety of lengths,either in their neutral (uncharged) forms or in forms which are salts,and either free of modifications such as glycosylation, side chainoxidation, phosphorylation, or any post-translational modification orcontaining these modifications, subject to the condition that themodification not destroy the biological activity of the polypeptides asherein described. In some embodiments, the neoantigenic peptides of thepresent disclosure can include: for MEW Class I, 22 residues or less inlength, e.g., from about 8 to about 22 residues, from about 8 to about15 residues, or 9 or 10 residues; for MEW Class II, 40 residues or lessin length, e.g., from about 8 to about 40 residues in length, from about8 to about 24 residues in length, from about 12 to about 19 residues, orfrom about 14 to about 18 residues. In some embodiments, a neoantigenicpeptide or neoantigenic polypeptide comprises a neoepitope.

The term “epitope” includes any protein determinant capable of specificbinding to an antibody, antibody peptide, and/or antibody-like molecule(including but not limited to a T cell receptor) as defined herein.Epitopic determinants typically consist of chemically active surfacegroups of molecules such as amino acids or sugar side chains andgenerally have specific three dimensional structural characteristics aswell as specific charge characteristics.

By “T-cell epitope” is meant a peptide sequence which can be bound bythe MEW molecules of class I or II in the form of a peptide-presentingMEW molecule or MEW complex and then, in this form, be recognized andbound by cytotoxic T-lymphocytes or T-helper cells, respectively.

The term “antibody” as used herein includes IgG (including IgG1, IgG2,IgG3, and IgG4), IgA (including IgA1 and IgA2), IgD, IgE, or IgM, andIgY, and is meant to include whole antibodies, including single-chainwhole antibodies, and antigen-binding (Fab) fragments thereof.Antigen-binding antibody fragments include, but are not limited to, Fab,Fab′ and F(ab′)₂, Fd (consisting of VH and CH1), single-chain variablefragment (scFv), single-chain antibodies, disulfide-linked variablefragment (dsFv) and fragments comprising either a VL or VH domain. Theantibodies can be from any animal origin. Antigen-binding antibodyfragments, including single-chain antibodies, can comprise the variableregion(s) alone or in combination with the entire or partial of thefollowing: hinge region, CH1, CH2, and CH3 domains. Also included areany combinations of variable region(s) and hinge region, CH1, CH2, andCH3 domains. Antibodies can be monoclonal, polyclonal, chimeric,humanized, and human monoclonal and polyclonal antibodies which, e.g.,specifically bind an HLA-associated polypeptide or an HLA-peptidecomplex. A person of skill in the art will recognize that a variety ofimmunoaffinity techniques are suitable to enrich soluble proteins, suchas soluble HLA-peptide complexes or membrane bound HLA-associatedpolypeptides, e.g., which have been proteolytically cleaved from themembrane. These include techniques in which (1) one or more antibodiescapable of specifically binding to the soluble protein are immobilizedto a fixed or mobile substrate—(e.g., plastic wells or resin, latex orparamagnetic beads), and (2) a solution containing the soluble proteinfrom a biological sample is passed over the antibody coated substrate,allowing the soluble protein to bind to the antibodies. The substratewith the antibody and bound soluble protein is separated from thesolution, and optionally the antibody and soluble protein aredisassociated, for example by varying the pH and/or the ionic strengthand/or ionic composition of the solution bathing the antibodies.Alternatively, immunoprecipitation techniques in which the antibody andsoluble protein are combined and allowed to form macromolecularaggregates can be used. The macromolecular aggregates can be separatedfrom the solution by size exclusion techniques or by centrifugation.

The term “immunopurification (IP)” (or immunoaffinity purification orimmunoprecipitation) is a process well known in the art and is widelyused for the isolation of a desired antigen from a sample. In general,the process involves contacting a sample containing a desired antigenwith an affinity matrix comprising an antibody to the antigen covalentlyattached to a solid phase. The antigen in the sample becomes bound tothe affinity matrix through an immunochemical bond. The affinity matrixis then washed to remove any unbound species. The antigen is removedfrom the affinity matrix by altering the chemical composition of asolution in contact with the affinity matrix. The immunopurification canbe conducted on a column containing the affinity matrix, in which casethe solution is an eluent. Alternatively the immunopurification can bein a batch process, in which case the affinity matrix is maintained as asuspension in the solution. An important step in the process is theremoval of antigen from the matrix. This is commonly achieved byincreasing the ionic strength of the solution in contact with theaffinity matrix, for example, by the addition of an inorganic salt. Analteration of pH can also be effective to dissociate the immunochemicalbond between antigen and the affinity matrix.

By “agent” is meant any small molecule chemical compound, antibody,nucleic acid molecule, or polypeptide, or fragments thereof.

By “alteration” or “change” is meant an increase or decrease. Analteration can be by as little as 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, orby 40%, 50%, 60%, or even by as much as 70%, 75%, 80%, 90%, or 100%.

By “biologic sample” is meant any tissue, cell, fluid, or other materialderived from an organism. As used herein, the term “sample” includes abiologic sample such as any tissue, cell, fluid, or other materialderived from an organism. By “specifically binds” is meant a compound(e.g., peptide) that recognizes and binds a molecule (e.g.,polypeptide), but which does not substantially recognize and bind othermolecules in a sample, for example, a biological sample.

By “capture reagent” is meant a reagent that specifically binds amolecule (e.g., a nucleic acid molecule or polypeptide) to select orisolate the molecule (e.g., a nucleic acid molecule or polypeptide).

As used herein, the terms “determining”, “assessing”, “assaying”,“measuring”, “detecting” and their grammatical equivalents refer to bothquantitative and qualitative determinations, and as such, the term“determining” is used interchangeably herein with “assaying,”“measuring,” and the like. Where a quantitative determination isintended, the phrase “determining an amount” of an analyte and the likeis used. Where a qualitative and/or quantitative determination isintended, the phrase “determining a level” of an analyte or “detecting”an analyte is used.

By “fragment” is meant a portion of a protein or nucleic acid that issubstantially identical to a reference protein or nucleic acid. In someembodiments, the portion retains at least 50%, 75%, or 80%, or 90%, 95%,or even 99% of the biological activity of the reference protein ornucleic acid described herein.

The terms “isolated,” “purified”, “biologically pure” and theirgrammatical equivalents refer to material that is free to varyingdegrees from components which normally accompany it as found in itsnative state. “Isolate” denotes a degree of separation from originalsource or surroundings. “Purify” denotes a degree of separation that ishigher than isolation. A “purified” or “biologically pure” protein issufficiently free of other materials such that any impurities do notmaterially affect the biological properties of the protein or causeother adverse consequences. That is, a nucleic acid or peptide of thepresent disclosure is purified if it is substantially free of cellularmaterial, viral material, or culture medium when produced by recombinantDNA techniques, or chemical precursors or other chemicals whenchemically synthesized. Purity and homogeneity are typically determinedusing analytical chemistry techniques, for example, polyacrylamide gelelectrophoresis or high performance liquid chromatography. The term“purified” can denote that a nucleic acid or protein gives rise toessentially one band in an electrophoretic gel. For a protein that canbe subjected to modifications, for example, phosphorylation orglycosylation, different modifications can give rise to differentisolated proteins, which can be separately purified.

By an “isolated” polypeptide (e.g., a peptide from a HLA-peptidecomplex) or polypeptide complex (e.g., a HLA-peptide complex) is meant apolypeptide or polypeptide complex of the present disclosure that hasbeen separated from components that naturally accompany it. Typically,the polypeptide or polypeptide complex is isolated when it is at least60%, by weight, free from the proteins and naturally-occurring organicmolecules with which it is naturally associated. The preparation can beat least 75%, at least 90%, or at least 99%, by weight, a polypeptide orpolypeptide complex of the present disclosure. An isolated polypeptideor polypeptide complex of the present disclosure can be obtained, forexample, by extraction from a natural source, by expression of arecombinant nucleic acid encoding such a polypeptide or one or morecomponents of a polypeptide complex, or by chemically synthesizing thepolypeptide or one or more components of the polypeptide complex. Puritycan be measured by any appropriate method, for example, columnchromatography, polyacrylamide gel electrophoresis, or by HPLC analysis.

The term “vectors” refers to a nucleic acid molecule capable oftransporting or mediating expression of a heterologous nucleic acid. Aplasmid is a species of the genus encompassed by the term “vector.” Avector typically refers to a nucleic acid sequence containing an originof replication and other entities necessary for replication and/ormaintenance in a host cell. Vectors capable of directing the expressionof genes and/or nucleic acid sequence to which they are operativelylinked are referred to herein as “expression vectors”. In general,expression vectors of utility are often in the form of “plasmids” whichrefer to circular double stranded DNA molecules which, in their vectorform are not bound to the chromosome, and typically comprise entitiesfor stable or transient expression or the encoded DNA. Other expressionvectors that can be used in the methods as disclosed herein include, butare not limited to plasmids, episomes, bacterial artificial chromosomes,yeast artificial chromosomes, bacteriophages or viral vectors, and suchvectors can integrate into the host's genome or replicate autonomouslyin the cell. A vector can be a DNA or RNA vector. Other forms ofexpression vectors known by those skilled in the art which serve theequivalent functions can also be used, for example, self-replicatingextrachromosomal vectors or vectors capable of integrating into a hostgenome. Exemplary vectors are those capable of autonomous replicationand/or expression of nucleic acids to which they are linked.

By “molecular profile” is meant a characterization of the expression orexpression level of two or more markers (e.g., polypeptides orpolynucleotides).

The terms “spacer” or “linker” as used in reference to a fusion proteinrefers to a peptide that joins the proteins comprising a fusion protein.Generally, a spacer has no specific biological activity other than tojoin or to preserve some minimum distance or other spatial relationshipbetween the proteins or RNA sequences. However, in some embodiments, theconstituent amino acids of a spacer can be selected to influence someproperty of the molecule such as the folding, net charge, orhydrophobicity of the molecule. Suitable linkers for use in anembodiment of the present disclosure are well known to those of skill inthe art and include, but are not limited to, straight or branched-chaincarbon linkers, heterocyclic carbon linkers, or peptide linkers. Thelinker is used to separate two antigenic peptides by a distancesufficient to ensure that, in some embodiments, each antigenic peptideproperly folds. Exemplary peptide linker sequences adopt a flexibleextended conformation and do not exhibit a propensity for developing anordered secondary structure. Typical amino acids in flexible proteinregions include Gly, Asn and Ser. Virtually any permutation of aminoacid sequences containing Gly, Asn and Ser would be expected to satisfythe above criteria for a linker sequence. Other near neutral aminoacids, such as Thr and Ala, also can be used in the linker sequence.Still other amino acid sequences that can be used as linkers aredisclosed in Maratea et al. (1985), Gene 40: 39-46; Murphy et al. (1986)Proc. Nat'l. Acad. Sci. USA 83: 8258-62; U.S. Pat. Nos. 4,935,233; and4,751,180.

The term “neoplasia” refers to any disease that is caused by or resultsin inappropriately high levels of cell division, inappropriately lowlevels of apoptosis, or both. Glioblastoma is one non-limiting exampleof a neoplasia or cancer. The terms “cancer” or “tumor” or“hyperproliferative disorder” refer to the presence of cells possessingcharacteristics typical of cancer-causing cells, such as uncontrolledproliferation, immortality, metastatic potential, rapid growth andproliferation rate, and certain characteristic morphological features.Cancer cells are often in the form of a tumor, but such cells can existalone within an animal, or can be a non-tumorigenic cancer cell, such asa leukemia cell. Cancers include, but are not limited to, B cell cancer,e.g., multiple myeloma, Waldenstrom's macroglobulinemia, the heavy chaindiseases, such as, for example, alpha chain disease, gamma chaindisease, and mu chain disease, benign monoclonal gammopathy, andimmunocytic amyloidosis, melanomas, breast cancer, lung cancer, bronchuscancer, colorectal cancer, prostate cancer (e.g., metastatic, hormonerefractory prostate cancer), pancreatic cancer, stomach cancer, ovariancancer, urinary bladder cancer, brain or central nervous system cancer,peripheral nervous system cancer, esophageal cancer, cervical cancer,uterine or endometrial cancer, cancer of the oral cavity or pharynx,liver cancer, kidney cancer, testicular cancer, biliary tract cancer,small bowel or appendix cancer, salivary gland cancer, thyroid glandcancer, adrenal gland cancer, osteosarcoma, chondrosarcoma, cancer ofhematological tissues, and the like. Other non-limiting examples oftypes of cancers applicable to the methods encompassed by the presentdisclosure include human sarcomas and carcinomas, e.g., fibrosarcoma,myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma,angiosarcoma, endotheliosarcoma, lymphangiosarcoma,lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor,leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, colorectal cancer,pancreatic cancer, breast cancer, ovarian cancer, squamous cellcarcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma,sebaceous gland carcinoma, papillary carcinoma, papillaryadenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogeniccarcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, livercancer, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor,cervical cancer, bone cancer, brain tumor, testicular cancer, lungcarcinoma, small cell lung carcinoma, bladder carcinoma, epithelialcarcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma,ependymoma, pinealoma, hemangioblastoma, acoustic neuroma,oligodendroglioma, meningioma, melanoma, neuroblastoma, retinoblastoma;leukemias, e.g., acute lymphocytic leukemia and acute myelocyticleukemia (myeloblastic, promyelocytic, myelomonocytic, monocytic anderythroleukemia); chronic leukemia (chronic myelocytic (granulocytic)leukemia and chronic lymphocytic leukemia); and polycythemia vera,lymphoma (Hodgkin's disease and non-Hodgkin's disease), multiplemyeloma, Waldenstrom's macroglobulinemia, and heavy chain disease. Insome embodiments, the cancer is an epithelial cancer such as, but notlimited to, bladder cancer, breast cancer, cervical cancer, coloncancer, gynecologic cancers, renal cancer, laryngeal cancer, lungcancer, oral cancer, head and neck cancer, ovarian cancer, pancreaticcancer, prostate cancer, or skin cancer. In other embodiments, thecancer is breast cancer, prostate cancer, lung cancer, or colon cancer.In still other embodiments, the epithelial cancer is non-small-cell lungcancer, nonpapillary renal cell carcinoma, cervical carcinoma, ovariancarcinoma (e.g., serous ovarian carcinoma), or breast carcinoma. Theepithelial cancers can be characterized in various other ways including,but not limited to, serous, endometrioid, mucinous, clear cell, brenner,or undifferentiated. In some embodiments, the present disclosure is usedin the treatment, diagnosis, and/or prognosis of lymphoma or itssubtypes, including, but not limited to, mantle cell lymphoma.Lymphoproliferative disorders are also considered to be proliferativediseases.

The term “vaccine” is to be understood as meaning a composition forgenerating immunity for the prophylaxis and/or treatment of diseases(e.g., neoplasia/tumor/infectious agents/autoimmune diseases).Accordingly, vaccines are medicaments which comprise antigens and areintended to be used in humans or animals for generating specific defenseand protective substance by vaccination. A “vaccine composition” caninclude a pharmaceutically acceptable excipient, carrier or diluent.Aspects of the present disclosure relate to use of the technology inpreparing an antigen-based vaccine. In these embodiments, vaccine ismeant to refer one or more disease-specific antigenic peptides (orcorresponding nucleic acids encoding them). In some embodiments, theantigen-based vaccine contains at least two, at least three, at leastfour, at least five, at least six, at least seven, at least eight, atleast nine, at least 10, at least 11, at least 12, at least 13, at least14, at least 15, at least 16, at least 17, at least 18, at least 19, atleast 20, at least 21, at least 22, at least 23, at least 24, at least25, at least 26, at least 27, at least 28, at least 29, at least 30, ormore antigenic peptides. In some embodiments, the antigen-based vaccinecontains from 2 to 100, 2 to 75, 2 to 50, 2 to 25, 2 to 20, 2 to 19, 2to 18, 2 to 17, 2 to 16, 2 to 15, 2 to 14, 2 to 13, 2 to 12, 2 to 10, 2to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, 3 to 100, 3 to 75, 3 to50, 3 to 25, 3 to 20, 3 to 19, 3 to 18, 3 to 17, 3 to 16, 3 to 15, 3 to14, 3 to 13, 3 to 12, 3 to 10, 3 to 9, 3 to 8, 3 to 7, 3 to 6, 3 to 5, 4to 100, 4 to 75, 4 to 50, 4 to 25, 4 to 20, 4 to 19, 4 to 18, 4 to 17, 4to 16, 4 to 15, 4 to 14, 4 to 13, 4 to 12, 4 to 10, 4 to 9, 4 to 8, 4 to7, 4 to 6, 5 to 100, 5 to 75, 5 to 50, 5 to 25, 5 to 20, 5 to 19, 5 to18, 5 to 17, 5 to 16, 5 to 15, 5 to 14, 5 to 13, 5 to 12, 5 to 10, 5 to9, 5 to 8, or 5 to 7 antigenic peptides. In some embodiments, theantigen-based vaccine contains 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, or 20 antigenic peptides. In some cases, theantigenic peptides are neoantigenic peptides. In some cases, theantigenic peptides comprise one or more neoepitopes.

The term “pharmaceutically acceptable” refers to approved or approvableby a regulatory agency of the Federal or a state government or listed inthe U.S. Pharmacopeia or other generally recognized pharmacopeia for usein animals, including humans. A “pharmaceutically acceptable excipient,carrier or diluent” refers to an excipient, carrier or diluent that canbe administered to a subject, together with an agent, and which does notdestroy the pharmacological activity thereof and is nontoxic whenadministered in doses sufficient to deliver a therapeutic amount of theagent. A “pharmaceutically acceptable salt” of pooled disease specificantigens as recited herein can be an acid or base salt that is generallyconsidered in the art to be suitable for use in contact with the tissuesof human beings or animals without excessive toxicity, irritation,allergic response, or other problem or complication. Such salts includemineral and organic acid salts of basic residues such as amines, as wellas alkali or organic salts of acidic residues such as carboxylic acids.Specific pharmaceutical salts include, but are not limited to, salts ofacids such as hydrochloric, phosphoric, hydrobromic, malic, glycolic,fumaric, sulfuric, sulfamic, sulfanilic, formic, toluene sulfonic,methane sulfonic, benzene sulfonic, ethane disulfonic,2-hydroxyethylsulfonic, nitric, benzoic, 2-acetoxybenzoic, citric,tartaric, lactic, stearic, salicylic, glutamic, ascorbic, pamoic,succinic, fumaric, maleic, propionic, hydroxymaleic, hydroiodic,phenylacetic, alkanoic such as acetic, HOOC—(CH2)n-COOH where n is 0-4,and the like. Similarly, pharmaceutically acceptable cations include,but are not limited to sodium, potassium, calcium, aluminum, lithium andammonium. Those of ordinary skill in the art will recognize from thisdisclosure and the knowledge in the art that further pharmaceuticallyacceptable salts for the pooled disease specific antigens providedherein, including those listed by Remington's Pharmaceutical Sciences,17th ed., Mack Publishing Company, Easton, Pa., p. 1418 (1985). Ingeneral, a pharmaceutically acceptable acid or base salt can besynthesized from a parent compound that contains a basic or acidicmoiety by any conventional chemical method. Briefly, such salts can beprepared by reacting the free acid or base forms of these compounds witha stoichiometric amount of the appropriate base or acid in anappropriate solvent.

Nucleic acid molecules useful in the methods of the disclosure includeany nucleic acid molecule that encodes a polypeptide of the disclosureor a fragment thereof. Such nucleic acid molecules need not be 100%identical with an endogenous nucleic acid sequence, but will typicallyexhibit substantial identity. Polynucleotides having substantialidentity to an endogenous sequence are typically capable of hybridizingwith at least one strand of a double-stranded nucleic acid molecule. By“hybridize” is meant pair to form a double-stranded molecule betweencomplementary polynucleotide sequences, or portions thereof, undervarious conditions of stringency. (See, e.g., Wahl, G. M. and S. L.Berger (1987) Methods Enzymol. 152:399; Kimmel, A. R. (1987) MethodsEnzymol. 152:507). For example, stringent salt concentration canordinarily be less than about 750 mM NaCl and 75 mM trisodium citrate,less than about 500 mM NaCl and 50 mM trisodium citrate, or less thanabout 250 mM NaCl and 25 mM trisodium citrate. Low stringencyhybridization can be obtained in the absence of organic solvent, e.g.,formamide, while high stringency hybridization can be obtained in thepresence of at least about 35% formamide, or at least about 50%formamide. Stringent temperature conditions can ordinarily includetemperatures of at least about 30° C., at least about 37° C., or atleast about 42° C. Varying additional parameters, such as hybridizationtime, the concentration of detergent, e.g., sodium dodecyl sulfate(SDS), and the inclusion or exclusion of carrier DNA, are well known tothose skilled in the art. Various levels of stringency are accomplishedby combining these various conditions as needed. In an exemplaryembodiment, hybridization can occur at 30° C. in 750 mM NaCl, 75 mMtrisodium citrate, and 1% SDS. In another exemplary embodiment,hybridization can occur at 37° C. in 500 mM NaCl, 50 mM trisodiumcitrate, 1% SDS, 35% formamide, and 100 μg/ml denatured salmon sperm DNA(ssDNA). In another exemplary embodiment, hybridization can occur at 42°C. in 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide, and200 μg/ml ssDNA. Useful variations on these conditions will be readilyapparent to those skilled in the art. For most applications, washingsteps that follow hybridization can also vary in stringency. Washstringency conditions can be defined by salt concentration and bytemperature. As above, wash stringency can be increased by decreasingsalt concentration or by increasing temperature. For example, stringentsalt concentration for the wash steps can be less than about 30 mM NaCland 3 mM trisodium citrate, or less than about 15 mM NaCl and 1.5 mMtrisodium citrate. Stringent temperature conditions for the wash stepscan include a temperature of at least about 25° C., of at least about42° C., or at least about 68° C. In exemplary embodiments, wash stepscan occur at 25° C. in 30 mM NaCl, 3 mM trisodium citrate, and 0.1% SDS.In other exemplary embodiments, wash steps can occur at 42° C. in 15 mMNaCl, 1.5 mM trisodium citrate, and 0.1% SDS. In another exemplaryembodiment, wash steps can occur at 68° C. in 15 mM NaCl, 1.5 mMtrisodium citrate, and 0.1% SDS. Additional variations on theseconditions will be readily apparent to those skilled in the art.Hybridization techniques are well known to those skilled in the art andare described, for example, in Benton and Davis (Science 196:180, 1977);Grunstein and Hogness (Proc. Natl. Acad. Sci., USA 72:3961, 1975);Ausubel et al. (Current Protocols in Molecular Biology, WileyInterscience, New York, 2001); Berger and Kimmel (Guide to MolecularCloning Techniques, 1987, Academic Press, New York); and Sambrook etal., Molecular Cloning: A Laboratory Manual, Cold Spring HarborLaboratory Press, New York.

By “substantially identical” is meant a polypeptide or nucleic acidmolecule exhibiting at least 50% identity to a reference amino acidsequence (for example, any one of the amino acid sequences describedherein) or nucleic acid sequence (for example, any one of the nucleicacid sequences described herein). Such a sequence can be at least 60%,80% or 85%, 90%, 95%, 96%, 97%, 98%, or even 99% or more identical atthe amino acid level or nucleic acid to the sequence used forcomparison. Sequence identity is typically measured using sequenceanalysis software (for example, Sequence Analysis Software Package ofthe Genetics Computer Group, University of Wisconsin BiotechnologyCenter, 1710 University Avenue, Madison, Wis. 53705, BLAST, BESTFIT,GAP, or PILEUP/PRETTYBOX programs). Such software matches identical orsimilar sequences by assigning degrees of homology to varioussubstitutions, deletions, and/or other modifications. Conservativesubstitutions typically include substitutions within the followinggroups: glycine, alanine; valine, isoleucine, leucine; aspartic acid,glutamic acid, asparagine, glutamine; serine, threonine; lysine,arginine; and phenylalanine, tyrosine. In an exemplary approach todetermining the degree of identity, a BLAST program can be used, with aprobability score between e-3 and e-m° indicating a closely relatedsequence. By “reference” is meant a standard of comparison.

The term “subject” or “patient” refers to an animal which is the objectof treatment, observation, or experiment. By way of example only, asubject includes, but is not limited to, a mammal, including, but notlimited to, a human or a non-human mammal, such as a non-human primate,murine, bovine, equine, canine, ovine, or feline.

The terms “treat,” “treated,” “treating,” “treatment,” and the like aremeant to refer to reducing, preventing, or ameliorating a disorderand/or symptoms associated therewith (e.g., a neoplasia or tumor orinfectious agent or an autoimmune disease). “Treating” can refer toadministration of the therapy to a subject after the onset, or suspectedonset, of a disease (e.g., cancer or infection by an infectious agent oran autoimmune disease). “Treating” includes the concepts of“alleviating”, which refers to lessening the frequency of occurrence orrecurrence, or the severity, of any symptoms or other ill effectsrelated to the disease and/or the side effects associated with therapy.The term “treating” also encompasses the concept of “managing” whichrefers to reducing the severity of a disease or disorder in a patient,e.g., extending the life or prolonging the survivability of a patientwith the disease, or delaying its recurrence, e.g., lengthening theperiod of remission in a patient who had suffered from the disease. Itis appreciated that, although not precluded, treating a disorder orcondition does not require that the disorder, condition, or symptomsassociated therewith be completely eliminated.

The term “prevent”, “preventing”, “prevention” and their grammaticalequivalents as used herein, means avoiding or delaying the onset ofsymptoms associated with a disease or condition in a subject that hasnot developed such symptoms at the time the administering of an agent orcompound commences.

The term “therapeutic effect” refers to some extent of relief of one ormore of the symptoms of a disorder (e.g., a neoplasia, tumor, orinfection by an infectious agent or an autoimmune disease) or itsassociated pathology. “Therapeutically effective amount” as used hereinrefers to an amount of an agent which is effective, upon single ormultiple dose administration to the cell or subject, in prolonging thesurvivability of the patient with such a disorder, reducing one or moresigns or symptoms of the disorder, preventing or delaying, and the likebeyond that expected in the absence of such treatment. “Therapeuticallyeffective amount” is intended to qualify the amount required to achievea therapeutic effect. A physician or veterinarian having ordinary skillin the art can readily determine and prescribe the “therapeuticallyeffective amount” (e.g., ED₅₀) of the pharmaceutical compositionrequired. For example, the physician or veterinarian can start doses ofthe compounds of the present disclosure employed in a pharmaceuticalcomposition at levels lower than that required in order to achieve thedesired therapeutic effect and gradually increase the dosage until thedesired effect is achieved. Disease, condition, and disorder are usedinterchangeably herein.

In some embodiments, the nucleic acid sequence encoding the HLA allelefurther comprises a peptide tag, an affinity tag, an epitope tag, or anaffinity acceptor tag which can be used to immunopurify the HLA-protein.Those of ordinary skill in the art will recognize that the terms“peptide tag,” “affinity tag,” “epitope tag,” or “affinity acceptor tag”are used interchangeably herein. As used herein, the term “affinityacceptor tag” refers to an amino acid sequence that permits the taggedprotein to be readily detected or purified, for example, by affinitypurification. An affinity acceptor tag is generally (but need not be)placed at or near the N- or C-terminus of a HLA allele. Various peptidetags are well known in the art. Non-limiting examples includepoly-histidine tag (e.g., 4 to 15 consecutive His residues, such as 8consecutive His residues); poly-histidine-glycine tag; HA tag (e.g.,Field et al., Mol. Cell. Biol., 8:2159, 1988); c-myc tag (e.g., Evans etal., Mol. Cell. Biol., 5:3610, 1985); Herpes simplex virus glycoproteinD (gD) tag (e.g., Paborsky et al., Protein Engineering, 3:547, 1990);FLAG tag (e.g., Hopp et al., BioTechnology, 6:1204, 1988; U.S. Pat. Nos.4,703,004 and 4,851,341); KT3 epitope tag (e.g., Martine et al.,Science, 255:192, 1992); tubulin epitope tag (e.g., Skinner, Biol.Chem., 266:15173, 1991); T7 gene 10 protein peptide tag (e.g.,Lutz-Freyemuth et al., Proc. Natl. Acad. Sci. USA, 87:6393, 1990);streptavidin tag (StrepTag™ or StrepTagII™; see, e.g., Schmidt et al.,J. Mol. Biol., 255(5):753-766, 1996 or U.S. Pat. No. 5,506,121; alsocommercially available from Sigma-Genosys); or a VSV-G epitope tagderived from the Vesicular Stomatis viral glycoprotein; or a V5 tagderived from a small epitope (Pk) found on the P and V proteins of theparamyxovirus of simian virus 5 (SV5). In some embodiments, the affinityacceptor tag is an “epitope tag,” which is a type of peptide tag thatadds a recognizable epitope (antibody binding site) to the HLA-proteinto provide binding of corresponding antibody, thereby allowingidentification or affinity purification of the tagged protein.Non-limiting example of an epitope tag is protein A or protein G, whichbinds to IgG. In some embodiments, the matrix of IgG Sepharose 6 FastFlow chromatography resin is covalently coupled to human IgG. This resinallows high flow rates, for rapid and convenient purification of aprotein tagged with protein A. Numerous other tag moieties are known to,and can be envisioned by, the ordinarily skilled artisan, and arecontemplated herein. Any peptide tag can be used as long as it iscapable of being expressed as an element of an affinity acceptor taggedHLA-peptide complex.

As used herein, the term “affinity molecule” refers to a molecule or aligand that binds with chemical specificity to an affinity acceptorpeptide. Chemical specificity is the ability of a protein's binding siteto bind specific ligands. The fewer ligands a protein can bind, thegreater its specificity. Specificity describes the strength of bindingbetween a given protein and ligand. This relationship can be describedby a dissociation constant (K_(D)), which characterizes the balancebetween bound and unbound states for the protein-ligand system.

The term “affinity acceptor tagged HLA-peptide complex” refers to acomplex comprising an HLA class I or class II-associated peptide or aportion thereof specifically bound to a single allelic recombinant classI or class II HLA peptide comprising an affinity acceptor peptide.

The terms “specific binding” or “specifically binding” when used inreference to the interaction of an affinity molecule and an affinityacceptor tag or an epitope and an HLA peptide means that the interactionis dependent upon the presence of a particular structure (i.e., theantigenic determinant or epitope) on the protein; in other words, theaffinity molecule is recognizing and binding to a specific affinityacceptor peptide structure rather than to proteins in general.

As used herein, the term “affinity” refers to a measure of the strengthof binding between two members of a binding pair, for example, an“affinity acceptor tag” and an “affinity molecule” and an HLA-bindingpeptide and a class I or II HLA. K_(D) is the dissociation constant andhas units of molarity. The affinity constant is the inverse of thedissociation constant. An affinity constant is sometimes used as ageneric term to describe this chemical entity. It is a direct measure ofthe energy of binding. Affinity can be determined experimentally, forexample by surface plasmon resonance (SPR) using commercially availableBiacore SPR units. Affinity can also be expressed as the inhibitoryconcentration 50 (IC₅₀), that concentration at which 50% of the peptideis displaced. Likewise, ln(IC₅₀) refers to the natural log of the IC₅₀.K_(off) refers to the off-rate constant, for example, for dissociationof an affinity molecule from the affinity acceptor tagged HLA-peptidecomplex.

In some embodiments, an affinity acceptor tagged HLA-peptide complexcomprises biotin acceptor peptide (BAP) and are immunopurified fromcomplex cellular mixtures using streptavidin/NeutrAvidin beads. Thebiotin-avidin/streptavidin binding is the strongest non-covalentinteraction known in nature. This property is exploited as a biologicaltool for a wide range of applications, such as immunopurification of aprotein to which biotin is covalently attached. In an exemplaryembodiment, the nucleic acid sequence encoding the HLA allele implementsbiotin acceptor peptide (BAP) as an affinity acceptor tag forimmunopurification. BAP can be specifically biotinylated in vivo or invitro at a single lysine residue within the tag (e.g., U.S. Pat. Nos.5,723,584; 5,874,239; and 5,932,433; and U.K Pat. No. GB2370039). BAP istypically 15 amino acids long and contains a single lysine as a biotinacceptor residue. In some embodiments, BAP is placed at or near the N-or C-terminus of a single allele HLA peptide. In some embodiments, BAPis placed in between a heavy chain domain and β2 microglobulin domain ofa class I HLA peptide. In some embodiments, BAP is placed in betweenβ-chain domain and α-chain domain of a class II HLA peptide. In someembodiments, BAP is placed in loop regions between α1, α2, and α3domains of the heavy chain of class I HLA, or between α1 and α2 and β1and β2 domains of the α-chain and β-chain, respectively of class II HLA.Exemplary constructs designed for HLA class I and II expressionimplementing BAP for biotinylation and immunopurification are describedin FIG. 2.

As used herein, the term “biotin” refers to the compound biotin itselfand analogues, derivatives and variants thereof. Thus, the term “biotin”includes biotin (cis-hexahydro-2-oxo-1H-thieno[3,4]imidazole-4-pentanoic acid) and any derivatives and analogsthereof, including biotin-like compounds. Such compounds include, forexample, biotin-e-N-lysine, biocytin hydrazide, amino or sulfhydrylderivatives of 2-iminobiotin and biotinyl-E-aminocaproicacid-N-hydroxysuccinimide ester, sulfosuccinimideiminobiotin,biotinbromoacetylhydrazide, p-diazobenzoyl biocytin,3-(N-maleimidopropionyl)biocytin, desthiobiotin, and the like. The term“biotin” also comprises biotin variants that can specifically bind toone or more of a Rhizavidin, avidin, streptavidin, tamavidin moiety, orother avidin-like peptides.

HLA Ligand Profiling Approaches

Biochemical peptide-MHC binding assay for HLA-epitope discovery was thebasis for NetMHC, the allele-specific predictor using artificial neuralnetworks; however, biochemical p:MHC binding assay slow is alow-throughput method (FIG. 19). Endogenously processed and presentedHLA-ligands profiled from cell lines and patient-derived materials arecommonly multi-allelic, meaning that LC-MS/MS data generated from thesesamples contain a mixed population of ligands that can bind to one ofthe multiple simultaneously expressed HLA alleles, as shown in FIG. 17and FIG. 19. Multi-allelic datasets require deconvolution to ascertainwhich peptides bind to the different HLA heterodimers presented by anindividual. Thus, ligands from multi-allelic datasets have to beassigned to their corresponding HLA heterodimers using either (1)binding predictors trained with preexisting data or (2) deconvolutionalgorithms that leverage overlap across HLA alleles represented in largeligand datasets. It is important to note that only LC-MS/MS datasetswith available HLA typing information can be confidently deconvoluted.In fact, nearly 40% of the naturally processed ligands bound to HLAclass I complexes reported from multi-allelic studies in the ImmuneEpitope Database (IEDB) lack HLA allele-specific assignments either dueto the lack of HLA typing information or inability to deconvolute,making it challenging to use this subset of data for allele-specificepitope prediction. In addition, it is difficult to identify peptidesbound to rare class I HLA heterodimers and many class II HLAheterodimers because there is not enough annotated data fordeconvolution. The multi-allelic data generation approach also limitsthe discovery of novel binding motifs as it deconvolution relies onpreexisting knowledge. Though there are caveats to utilizingmulti-allelic datasets for allele-specific epitope predictions, they areimmensely valuable for determining patterns of ligand presentation thatrequire co-expression of multiple alleles and for validating epitopeprediction algorithms.

An orthogonal approach to multi-allelic data generation and subsequentdeconvolution is the creation of mono-allelic datasets from whichpeptide populations presented by a single HLA allele are identified(FIG. 17 and FIG. 19). One method for generating mono-allelic datautilizes cell lines that are deficient in HLA expression. These cellscan be transfected or transduced with single HLA alleles, so ligands canbe profiled by LC-MS/MS to generate allele-specific ligand libraries.Peptides bound to soluble HLA (sHLA) molecules can also be isolated fromcell media and profiled by LC-MS/MS to produce mono-allelic data. Amajor advantage of mono-allelic datasets is that they require nodeconvolution and enable confident peptide-HLA allele assignmentswithout preexisting data. Mono-allelic approaches also rapidly providedata for HLA alleles that have not been characterized previously—a taskthat multi-allelic data can do only if enough overlap is present amongstlarge datasets. Additionally, novel peptide-binding motifs can easily bediscovered using mono-allelic systems as no previous knowledge isrequired for confident HLA-binding assignments. Mono-allelic data caneven be leveraged to assign ligands from multi-allelic datasets whendeconvolution methods fail to do so.

The limiting factor of currently available mono-allelic approach is thatit requires an HLA deficient cell line. A key innovative feature of thepresent disclosure is that an HLA deficient cell line is not requiredfor mono-allelic data generation. The affinity-tagged constructs asprovided herein can be put into any cell line presenting endogenousHLA-peptide complexes to isolate the allele of interest using theaffinity tag. Another advantage of the present disclosure is that thesame reagents can be used for any class I or class II allele in thelibrary provided that it has the same affinity tag, making presentlydisclosed method scalable (automated). In some embodiments, the methodcomprises expressing a library of peptides in the population of cells,thereby forming a library of affinity acceptor tagged HLA-peptidecomplexes. In some embodiments, the method comprises contacting to thepopulation of cells a library of peptides or a library of sequencesencoding peptides, thereby forming a library of affinity acceptor taggedHLA-peptide complexes. In some embodiments, the library comprises alibrary of peptides associated with a disease or condition. In someembodiments, the disease or condition is cancer. In some embodiments,the population of cells is from a biological sample from a subject witha disease or condition.

In some embodiments, the method further comprises isolating the peptidesfrom the affinity acceptor tagged HLA-peptide complexes before thecharacterizing. In some embodiments, the peptides are isolated usinganti-HLA antibodies. In some cases, soluble HLA (sHLA) with affinitytags are isolated using anti-HLA antibodies. In some cases, soluble HLA(sHLA) with affinity tags are isolated using a column containing ananti-HLA antibody.

Methods and Compositions

Provided herein is a method of characterizing HLA-peptide complexescomprising: providing a population of cells, wherein one or more cellsof the population of cells comprise a polynucleic acid comprising asequence encoding an affinity acceptor tagged class I or class II HLAallele, wherein the sequence encoding an affinity acceptor tagged HLAcomprises a sequence encoding a recombinant class I or class II HLAallele operatively linked to a sequence encoding an affinity acceptorpeptide; expressing the affinity acceptor tagged HLA in at least onecell of the one or more cells of the population of cells, therebyforming affinity acceptor tagged HLA-peptide complexes in the at leastone cell; enriching for the affinity acceptor tagged HLA-peptidecomplexes; and characterizing HLA-peptide complexes.

In some embodiments, the characterizing comprises characterizing apeptide from the affinity acceptor tagged HLA-peptide complex. In someembodiments, the method comprises carrying out the steps of the methodfor different class I and/or class II HLA alleles. In some embodiments,the method comprises using more than one class I and/or class II HLAallele. In some embodiments, the population of cells are derived from asubject (e.g., a patient having a disease). In some embodiments, thepopulation of cells are class I and/or class II negative cell lines. Insome embodiments, the method further comprises generating an HLA-allelespecific peptide database.

Provided herein is a method of generating an HLA-allele specific peptidedatabase comprising: providing a first and a second population of cellseach comprising one or more cells comprising an affinity acceptor taggedHLA, wherein the sequence affinity acceptor tagged HLA comprises adifferent recombinant polypeptide encoded by a different HLA alleleoperatively linked to an affinity acceptor peptide; enriching foraffinity acceptor tagged HLA-peptide complexes; characterizing a peptideor a portion thereof bound to an affinity acceptor tagged HLA-peptidecomplex from the enriching; and generating an HLA-allele specificpeptide database.

In some embodiments, the enriching does not comprise use of a tetramerreagent.

In some embodiments, the characterizing comprises determining thesequence of a peptide or a portion thereof bound to an affinity acceptortagged HLA-peptide complex from the enriching. In some embodiments, thecharacterizing comprises determining whether the peptide or a portionthereof is modified (e.g., post-translational modification). In someembodiments, the determining comprises biochemical analysis. In someembodiments, the determining comprises mass spectrometry analysis. Insome embodiments, the mass spectrometry is MS analysis, MS/MS analysis,LC-MS/MS analysis, or a combination thereof. In some embodiments, MSanalysis is used to determine a mass of an intact peptide. For example,the determining can comprise determining a mass of an intact peptide(e.g., MS analysis). In some embodiments, MS/MS analysis is used todetermine a mass of peptide fragments. For example, the determining cancomprise determining a mass of peptide fragments, which can be used todetermine an amino acid sequence of a peptide or portion thereof (e.g.,MS/MS analysis). In some embodiments, the mass of peptide fragments isused to determine a sequence of amino acids within the peptide. In someembodiments, LC-MS/MS analysis used to separate complex peptidemixtures. For example, the determining can comprise separating complexpeptide mixtures, such as by liquid chromatography, and determining amass of an intact peptide, a mass of peptide fragments, or a combinationthereof (e.g., LC-MS/MS analysis). This data can be used, e.g., forpeptide sequencing.

In some embodiments, the characterizing comprises evaluating a bindingaffinity or stability of a peptide or a portion thereof bound to anaffinity acceptor tagged HLA-peptide complex from the enriching. In someembodiments, the characterizing comprises determining whether a peptideor a portion thereof bound to an affinity acceptor tagged HLA-peptidecomplex from the enriching contains one or more mutations. In someembodiments, the characterizing comprises determining whether thepeptide or a portion thereof is modified (e.g., post-translationalmodification). In some embodiments, the characterizing comprisesevaluating associations of peptides of affinity acceptor taggedHLA-peptide complexes with HLA alleles.

In some embodiments, the method comprises expressing a library ofpeptides in the population of cells, thereby forming a library ofaffinity acceptor tagged HLA-peptide complexes. In some embodiments, themethod comprises contacting to the population of cells a library ofpeptides or a library of sequences encoding peptides, thereby forming alibrary of affinity acceptor tagged HLA-peptide complexes. In someembodiments, the library comprises a library of peptides associated witha disease or condition. In some embodiments, the disease or condition iscancer. In some embodiments, the population of cells is from abiological sample from a subject with a disease or condition.

In some embodiments, the population of cells is a cell line. In someembodiments, the population of cells is a population of primary cells.

In some embodiments, the recombinant class I or class II HLA allele ismatched to a subject with a disease or condition. In some embodiments,an antigen presenting cell comprising the peptide or a mutant thereofbound to an affinity acceptor tagged HLA-peptide complex has reactivityto a T cell expressing a T cell receptor from a subject. In someembodiments, the characterizing comprises comparing HLA-peptidecomplexes from cancer cells to HLA-peptide complexes from non-cancercells.

In some embodiments, the population of cells is a knock-out of one ormore HLA class I alleles. In some embodiments, the population of cellsis a knock-out of one or more HLA class II alleles. In some embodiments,the population of cells is a knock-out of all HLA class I alleles. Insome embodiments, the population of cells is a knock-out of all HLAclass II alleles. In some embodiments, the population of cells is aknock-out of all HLA class I alleles and a knock-out of all HLA class IIalleles. In some embodiments, knock-out of an HLA class I or class IIallele comprises elimination of the function of the HLA class I or classII allele. In some embodiments, knock-out of the HLA class I or class IIallele is achieved through gene editing. In some embodiments, geneediting is performed by administering to an individual in need thereof anuclease, wherein the nuclease targets the HLA class I allele or classII allele to be knocked-out. In some embodiments, the nuclease is aCRISPR associated protein (e.g. Cas proteins, e.g., Cas9), a Zinc fingernuclease (ZFN), a Transcription Activator-Like Effector Nuclease(TALEN), or a meganuclease. In some embodiments, gene editing isachieved by administering to an individual in need thereof a CRISPR-Cas9system. In some embodiments, any suitable nuclease that induces a nickor double-stranded break into a desired recognition site is used. Insome embodiments, a naturally-occurring or native nuclease is used. Insome embodiments, a modified or engineered nuclease is used.

In some embodiments, the population of cells is a knock-down of one ormore HLA class I alleles. In some embodiments, the population of cellsis a knock-down of one or more HLA class II alleles. In someembodiments, the population of cells is a knock-down of all HLA class Ialleles. In some embodiments, the population of cells is a knock-down ofall HLA class II alleles. In some embodiments, the population of cellsis a knock-down of all HLA class I alleles and a knock-out of all HLAclass II alleles. In some embodiments, knock-down of an HLA class I orclass II allele comprises a reduction in the expression of the HLA classI or class II allele. In some embodiments, knock-down of the HLA class Iallele or class II allele is achieved by administering to an individualin need thereof a therapeutically effective amount of a smalldouble-stranded interfering RNA (siRNA), a microRNA (miRNA), a shorthairpin RNA (shRNA), wherein the siRNA, miRNA, shRNA targets the HLAclass I allele or class II allele to be knocked-down. In someembodiments, the expression of the HLA class I or class II allele isreduced by about 99%, about 95%, about 90%, about 85%, about 80%, about75%, about 70%, about 65%, about 60%, about 55%, about 50%, about 45%,about 40%, about 35%, about 30%, about 25%, or about 20% compared towhen the HLA class I allele or class II allele has not beenknocked-down.

In some embodiments, the population of cells comprises cells that havebeen enriched or sorted for cell surface expression of an HLA class Iallele, an HLA class II allele, or a combination thereof, such as byfluorescence activated cell sorting (FACS). In some embodiments,fluorescence activated cell sorting (FACS) is used to sort thepopulation of cells. In some embodiments, fluorescence activated cellsorting (FACS) is used to sort the population of cells for cell surfaceexpression of an HLA class I allele, an HLA class II allele, or acombination thereof. In some embodiments, FACS is used to enrich or sortfor low cell surface HLA class I or class II expressing cells.

In some embodiments, the population of cells comprises a plurality ofpopulations of cells, each expressing a different recombinant class I orclass II HLA allele. In some embodiments, each population of cells ofthe plurality is in a separate container.

In some embodiments, the method further comprises isolating peptidesfrom the affinity acceptor tagged HLA-peptide complexes before thecharacterizing. In some embodiments, the method further comprisestrimming a terminus of the peptide bound to the HLA-peptide complexes(FIG. 13).

In some embodiments, the population of cells expresses one or moreendogenous HLA alleles. In some embodiments, the population of cells isan engineered population of cells lacking one or more endogenous HLAclass I alleles. In some embodiments, the population of cells is anengineered population of cells lacking endogenous HLA class I alleles.In some embodiments, the population of cells is an engineered populationof cells lacking one or more endogenous HLA class II alleles. In someembodiments, the population of cells is an engineered population ofcells lacking endogenous HLA class II alleles. In some embodiments, thepopulation of cells is an engineered population of cells lackingendogenous HLA class I alleles and endogenous HLA class II alleles. Insome embodiments, the sequence encoding a recombinant class I or classII HLA allele encodes a class I HLA. In some embodiments, the sequenceencoding a recombinant class I or class II HLA allele encodes a class IIHLA. In some embodiments, the class I HLA is selected from the groupconsisting of HLA-A, HLA-B, HLA-C. In some embodiments, the class I HLAis a non-classical class-I-b group. In some embodiments, the class I HLAis selected from the group consisting of HLA-E, HLA-F, and HLA-G. Insome embodiments, the class I HLA is a non-classical class-I-b groupselected from the group consisting of HLA-E, HLA-F, and HLA-G. In someembodiments, the class II HLA comprises a HLA class II α-chain, a HLAclass II β-chain, or a combination thereof.

In some embodiments, each sequence encoding a different class I and/orclass II HLA allele is operatively linked to a sequence encoding adifferent affinity acceptor peptide. In some embodiments, the sequenceencoding an affinity acceptor peptide is operatively linked to asequence encoding a recombinant class I or class II HLA allele thatencodes for an extracellular portion of the recombinant class I or classII HLA allele. In some embodiments, the encoded affinity acceptorpeptide is expressed extracellularly. In some embodiments, the sequenceencoding an affinity acceptor peptide is operatively linked to theN-terminus of the sequence encoding a recombinant class I or class IIHLA allele. In some embodiments, the sequence encoding an affinityacceptor peptide is operatively linked to a sequence encoding arecombinant class I or class II HLA allele that encodes for anintracellular portion of the recombinant class I or class II HLA allele.In some embodiments, the encoded affinity acceptor peptide is expressedintracellularly. In some embodiments, the sequence encoding an affinityacceptor peptide is operatively linked to the C-terminus of the sequenceencoding a recombinant class I or class II HLA allele.

In some embodiments, the sequence encoding an affinity acceptor peptideis operatively linked to the sequence encoding a recombinant class I orclass II HLA allele by a linker.

In some embodiments, the enriching comprises enriching for intact cellsexpressing the affinity acceptor tagged HLA-peptide complexes.

In some embodiments, the method does not comprise lysing the one or morecells before the enriching. In some embodiments, the method furthercomprises lysing the one or more cells before the enriching.

In some embodiments, the enriching comprises contacting an affinityacceptor peptide binding molecule to the affinity acceptor taggedHLA-peptide complexes, wherein the affinity acceptor peptide bindingmolecule binds specifically to the affinity acceptor peptide. In someembodiments, the affinity acceptor peptide can comprise a biotinacceptor peptide (BAP), poly-histidine tag, poly-histidine-glycine tag,poly-arginine tag, poly-aspartate tag, poly-cysteine tag,poly-phenylalanine, c-myc tag, Herpes simplex virus glycoprotein D (gD)tag, FLAG tag, KT3 epitope tag, tubulin epitope tag, T7 gene 10 proteinpeptide tag, streptavidin tag, streptavidin binding peptide (SPB) tag,Strep-tag, Strep-tag II, albumin-binding protein (ABP) tag, alkalinephosphatase (AP) tag, bluetongue virus tag (B-tag), calmodulin bindingpeptide (CBP) tag, chloramphenicol acetyl transferase (CAT) tag,choline-binding domain (CBD) tag, chitin binding domain (CBD) tag,cellulose binding domain (CBP) tag, dihydrofolate reductase (DHFR) tag,galactose-binding protein (GBP) tag, maltose binding protein (MBP),glutathione-S-transferase (GST), Glu-Glu (EE) tag, human influenzahemagglutinin (HA) tag, horseradish peroxidase (HRP) tag, NE-tag, HSVtag, ketosteroid isomerase (KSI) tag, KT3 tag, LacZ tag, luciferase tag,NusA tag, PDZ domain tag, AviTag, Calmodulin-tag, E-tag, S-tag, SBP-tag,Softag 1, Softag 3, TC tag, VSV-tag, Xpress tag, Isopeptag, SpyTag,SnoopTag, Profinity eXact tag, Protein C tag, S1-tag, S-tag,biotin-carboxy carrier protein (BCCP) tag, green fluorescent protein(GFP) tag, small ubiquitin-like modifier (SUMO) tag, tandem affinitypurification (TAP) tag, HaloTag, Nus-tag, Thioredoxin-tag, Fc-tag, CYDtag, HPC tag, TrpE tag, ubiquitin tag, VSV-G epitope tag, V5 tag, or acombination thereof; optionally, wherein the affinity acceptor peptidecomprises two or more repeats of a tag sequence. In some embodiments,the affinity acceptor peptide binding molecule is biotin or an antibodyspecific to the affinity acceptor peptide.

In some embodiments, the enriching comprises contacting an affinitymolecule to the affinity acceptor tagged HLA-peptide complexes, whereinthe affinity molecule binds specifically to the affinity acceptorpeptide binding molecule. In some embodiments, the affinity molecule isstreptavidin, NeutrAvidin, or a derivative thereof. In some embodiments,the enriching comprises immunoprecipitating affinity acceptor taggedHLA-peptide complexes. In some embodiments, the affinity acceptorpeptide binding molecule is attached to a solid surface. In someembodiments, the affinity molecule is attached to a solid surface. Insome embodiments, the solid surface is a bead.

In some embodiments, the enriching comprises immunoprecipitatingaffinity acceptor tagged HLA-peptide complexes with an affinity acceptorpeptide binding molecule that binds specifically to the affinityacceptor peptide. In some embodiments, the affinity acceptor peptidebinding molecule does not specifically interact with the amino acidsequence of the encoded recombinant class I or class II HLA. In someembodiments, the enriching comprises contacting an affinity moleculespecific to an extracellular portion of the HLA-peptide complexes. Insome embodiments, the enriching comprises contacting an affinitymolecule specific to an N-terminal portion of the HLA-peptide complexes.

In some embodiments, the providing comprises contacting the populationof cells with the polynucleic acid comprising a sequence encoding anaffinity acceptor tagged HLA. In some embodiments, the contactingcomprises transfecting or transducing. In some embodiments, theproviding comprises contacting the population of cells with a vector orplasmid comprising the polynucleic acid comprising a sequence encodingan affinity acceptor tagged HLA. In some embodiments, the vector is aviral vector.

Any suitable biochemical assay can be used to determine an HLA expressedin a cell (e.g., an engineered cell line). Exemplary methods todetermine the identity of an HLA allele expressed in a cell (e.g., anengineered cell line) include Western blot analysis, e.g., to determinethe class of an HLA allele (class I or class II), sequence analysis,e.g., sequencing individual alleles (e.g., using different primers foridentification of different alleles of similar sequence). In someembodiments, a polynucleic acid encoding a HLA allele comprises abarcode sequence. The barcode sequence can be used to identify an HLAallele expressed in a cell. In some embodiments, the barcode sequence isunique to a single HLA. In some embodiments, the barcode sequence isunique to a single HLA class I or class II allele.

In some embodiments, the polynucleic acid comprising a sequence encodingan affinity acceptor tagged HLA is stably integrated into the genome ofthe population of cells. In some embodiments, sequence encoding arecombinant class I or class II HLA comprises a sequence encoding a HLAclass I α-chain. In some embodiments, the method further comprisesexpressing a sequence encoding β2 microglobulin in the one or morecells. In some embodiments, the sequence encoding β2 microglobulin isconnected to the sequence encoding a HLA class I α-chain. In someembodiments, the sequence encoding β2 microglobulin is connected to thesequence encoding a HLA class I α-chain by a linker. In someembodiments, the sequence encoding β2 microglobulin is connected to asequence encoding a second affinity acceptor peptide.

In some embodiments, the sequence encoding a recombinant class I orclass II HLA comprises a sequence encoding a HLA class II α-chain. Insome embodiments, the method further comprises expressing a sequenceencoding a HLA class II β-chain in the one or more cells. In someembodiments, the sequence encoding a HLA class II β-chain is connectedto the sequence encoding a HLA class II α-chain. In some embodiments,the sequence encoding a HLA class II β-chain is connected to thesequence encoding a HLA class II α-chain by a linker. In someembodiments, the sequence encoding a HLA class II β-chain is connectedto a sequence encoding a second affinity acceptor peptide.

In some embodiments, the second affinity acceptor peptide is differentthan the first affinity acceptor peptide and can comprise a biotinacceptor peptide (BAP), poly-histidine tag, poly-histidine-glycine tag,poly-arginine tag, poly-aspartate tag, poly-cysteine tag,poly-phenylalanine, c-myc tag, Herpes simplex virus glycoprotein D (gD)tag, FLAG tag, KT3 epitope tag, tubulin epitope tag, T7 gene 10 proteinpeptide tag, streptavidin tag, streptavidin binding peptide (SPB) tag,Strep-tag, Strep-tag II, albumin-binding protein (ABP) tag, alkalinephosphatase (AP) tag, bluetongue virus tag (B-tag), calmodulin bindingpeptide (CBP) tag, chloramphenicol acetyl transferase (CAT) tag,choline-binding domain (CBD) tag, chitin binding domain (CBD) tag,cellulose binding domain (CBP) tag, dihydrofolate reductase (DHFR) tag,galactose-binding protein (GBP) tag, maltose binding protein (MBP),glutathione-S-transferase (GST), Glu-Glu (EE) tag, human influenzahemagglutinin (HA) tag, horseradish peroxidase (HRP) tag, NE-tag, HSVtag, ketosteroid isomerase (KSI) tag, KT3 tag, LacZ tag, luciferase tag,NusA tag, PDZ domain tag, AviTag, Calmodulin-tag, E-tag, S-tag, SBP-tag,Softag 1, Softag 3, TC tag, VSV-tag, Xpress tag, Isopeptag, SpyTag,SnoopTag, Profinity eXact tag, Protein C tag, S1-tag, S-tag,biotin-carboxy carrier protein (BCCP) tag, green fluorescent protein(GFP) tag, small ubiquitin-like modifier (SUMO) tag, tandem affinitypurification (TAP) tag, HaloTag, Nus-tag, Thioredoxin-tag, Fc-tag, CYDtag, HPC tag, TrpE tag, ubiquitin tag, VSV-G epitope tag, V5 tag, or acombination thereof.

In some embodiments, the linker comprises a polynucleic acid sequenceencoding a cleavable linker. In some embodiments, the cleavable linkeris a ribosomal skipping site or an internal ribosomal entry site (IRES)element. In some embodiments, the ribosomal skipping site or IRES iscleaved when expressed in the cells. In some embodiments, the ribosomalskipping site is selected from the group consisting of F2A, T2A, P2A,and E2A. In some embodiments, the IRES element is selected from commoncellular or viral IRES sequences.

In some embodiments, the determining comprises performing massspectrometry, such as tandem mass spectrometry. In some embodiments, thedetermining comprises obtaining a peptide sequence that corresponds toan MS/MS spectra of one or more peptides isolated from the enrichedaffinity acceptor tagged HLA-peptide complexes from a peptide database;wherein one or more sequences obtained identifies the sequence of theone or more peptides.

In some embodiments, the population of cells is a cell line is selectedfrom HEK293T, expi293, HeLa, A375, 721.221, JEG-3, K562, Jurkat, Hep G2,SH-SY5Y, CACO-2, U937, U-2 OS, ExpiCHO, CHO and THP1. In someembodiments, the cell line is treated with one or more cytokines,checkpoint inhibitors, epigenetically-active drugs, IFN-γ, an agent thatalters antigen processing (e.g., peptidase inhibitors, proteasomeinhibitor, TAP inhibitor, etc.), or a combination thereof. In someembodiments, the peptide database is a no-enzyme specificity peptidedatabase, such as a without modification database or a with modification(e.g., phosphorylation or cysteinylation) database. In some embodiments,the peptide database is a polypeptide database. In some embodiments, thepolypeptide database is a protein database. In some embodiments, themethod further comprises searching the peptide database using areversed-database search strategy. In some embodiments, the methodfurther comprises searching a protein database using a reversed-databasesearch strategy. In some embodiments, a de novo search is performed,e.g., to discover new peptides that are not included in a normal peptideor protein database.

In some embodiments, the population of cells comprises at least 10⁵cells, at least 10⁶ cells or at least 10⁷ cells. In some embodiments,the population of cells is a population of dendritic cells, macrophages,cancer cells or B-cells. In some embodiments, the population of cellscomprises tumor cells or cells infected by an infectious agent or aportion thereof.

In some embodiments, the population of cells is contacted with an agentprior to isolating said HLA-peptide complexes from the one or morecells. In some embodiments, said agent is an inflammatory cytokine, achemical agent, an adjuvant, a therapeutic agent or radiation.

In some embodiments, the HLA allele is a mutated HLA allele.

In some embodiments, the method comprises carrying out the steps of themethod for different HLA alleles.

Provided herein is a HLA-allele specific binding peptide sequencedatabase obtained by carrying out the methods described herein. Providedherein is a combination of two or more HLA-allele specific bindingpeptide sequence databases obtained by carrying out the methodsdescribed herein repeatedly, each time using a different HLA-allele.Provided herein is a method for generating a prediction algorithm foridentifying HLA-allele specific binding peptides, comprising training amachine with a peptide sequence database of described herein. In someembodiments, the machine combines one or more linear models, supportvector machines, decision trees and neural networks.

Generating a prediction algorithm by training a machine is a well-knowntechnique. The most important in the training of the machine is thequality of the database used for the training. Typically, the machinecombines one or more linear models, support vector machines, decisiontrees and/or a neural network.

In some embodiments, a variable used to train the machine or algorithmcomprises one or more variables selected from the group consisting ofpeptide sequence, amino acid physical properties, peptide physicalproperties, expression level of the source protein of a peptide within acell, protein stability, protein translation rate, ubiquitination sites,protein degradation rate, translational efficiencies from ribosomalprofiling, protein cleavability, protein localization, motifs of hostprotein that facilitate TAP transport, host protein is subject toautophagy, motifs that favor ribosomal stalling (e.g., polyproline orpolylysine stretches), protein features that favor NMD (e.g., long 3′UTR, stop codon >50nt upstream of last exon:exon junction and peptidecleavability).

Provided herein is a method for identifying HLA-allele specific bindingpeptides comprising analyzing the sequence of a peptide with a machinewhich has been trained with a peptide sequence database obtained bycarrying out a method described herein for the HLA-allele. In someembodiments, the method comprises determining the expression level ofthe source protein of the peptide within a cell; and wherein the sourceprotein expression is a predictive variable used by the machine. In someembodiments, the expression level is determined by measuring the amountof source protein or the amount of RNA encoding said source protein.

Provided herein is a composition comprising a first and a secondrecombinant polynucleic acid each comprising a sequence encoding anaffinity acceptor tagged HLA, wherein the sequence encoding an affinityacceptor tagged HLA comprises (a) a sequence encoding a differentrecombinant HLA class I α-chain allele, (b) a sequence encoding anaffinity acceptor peptide, and optionally, (c) a sequence encoding β2microglobulin; wherein the sequences of (a) and (b), and optionally (c),are operatively linked.

Provided herein is a composition comprising a first and a secondrecombinant polynucleic acid each comprising a sequence encoding anaffinity acceptor tagged HLA, wherein the sequence encoding an affinityacceptor tagged HLA comprises (a) a sequence encoding a recombinant HLAclass II α-chain allele, (b) a sequence encoding an affinity acceptorpeptide, and optionally, (c) a sequence encoding a HLA class II β-chain;wherein the sequences of (a) and (b), and optionally (c), areoperatively linked.

In some embodiments, the first and second recombinant polynucleic acidsare isolated.

In some embodiments, the sequence encodes a recombinant class I or classII HLA allele. In some embodiments, the class I HLA is selected from thegroup consisting of HLA-A, HLA-B, HLA-C. In some embodiments, the classI HLA is a non-classical class-I-b group. In some embodiments, the classI HLA is selected from the group consisting of HLA-E, HLA-F, and HLA-G.In some embodiments, the class I HLA is a non-classical class-I-b groupselected from the group consisting of HLA-E, HLA-F, and HLA-G.

In some embodiments, for both the first and the second recombinantpolynucleic acids: the sequence encoding an affinity acceptor peptide isoperatively linked to a sequence of the sequence encoding a differentrecombinant HLA allele that encodes for an extracellular portion of thedifferent recombinant HLA allele. In some embodiments, for both thefirst and the second recombinant polynucleic acids: the sequenceencoding an affinity acceptor molecule is operatively linked to theN-terminus of the sequence encoding a different recombinant HLA allele.In some embodiments, for both the first and the second recombinantpolynucleic acids: the sequence encoding an affinity acceptor peptide isoperatively linked to a sequence of the sequence encoding a differentrecombinant HLA allele that encodes for an intracellular portion of thedifferent recombinant HLA allele. In some embodiments, for both thefirst and the second recombinant polynucleic acids: the sequenceencoding an affinity acceptor peptide is operatively linked to theC-terminus of the sequence encoding a different recombinant HLA allele.In some embodiments, for both the first and the second recombinantpolynucleic acids: the sequence encoding an affinity acceptor peptide isoperatively linked to the sequence encoding a different recombinant HLAallele by a linker. In some embodiments, the encoded affinity acceptorpeptide binds specifically to an affinity acceptor peptide bindingmolecule. In some embodiments, the affinity acceptor peptide of thefirst and the second recombinant polynucleic acids is different.

In some embodiments, the encoded affinity acceptor peptide can comprisea biotin acceptor peptide (BAP), poly-histidine tag,poly-histidine-glycine tag, poly-arginine tag, poly-aspartate tag,poly-cysteine tag, poly-phenylalanine, c-myc tag, Herpes simplex virusglycoprotein D (gD) tag, FLAG tag, KT3 epitope tag, tubulin epitope tag,T7 gene 10 protein peptide tag, streptavidin tag, streptavidin bindingpeptide (SPB) tag, Strep-tag, Strep-tag II, albumin-binding protein(ABP) tag, alkaline phosphatase (AP) tag, bluetongue virus tag (B-tag),calmodulin binding peptide (CBP) tag, chloramphenicol acetyl transferase(CAT) tag, choline-binding domain (CBD) tag, chitin binding domain (CBD)tag, cellulose binding domain (CBP) tag, dihydrofolate reductase (DHFR)tag, galactose-binding protein (GBP) tag, maltose binding protein (MBP),glutathione-S-transferase (GST), Glu-Glu (EE) tag, human influenzahemagglutinin (HA) tag, horseradish peroxidase (HRP) tag, NE-tag, HSVtag, ketosteroid isomerase (KSI) tag, KT3 tag, LacZ tag, luciferase tag,NusA tag, PDZ domain tag, AviTag, Calmodulin-tag, E-tag, S-tag, SBP-tag,Softag 1, Softag 3, TC tag, VSV-tag, Xpress tag, Isopeptag, SpyTag,SnoopTag, Profinity eXact tag, Protein C tag, S1-tag, S-tag,biotin-carboxy carrier protein (BCCP) tag, green fluorescent protein(GFP) tag, small ubiquitin-like modifier (SUMO) tag, tandem affinitypurification (TAP) tag, HaloTag, Nus-tag, Thioredoxin-tag, Fc-tag, CYDtag, HPC tag, TrpE tag, ubiquitin tag, VSV-G epitope tag, V5 tag, or acombination thereof; optionally, wherein the affinity acceptor peptidecomprises two or more repeats of a tag sequence. In some embodiments,the affinity acceptor peptide binding molecule is biotin or an antibodyspecific to the affinity acceptor peptide. In some embodiments, theaffinity acceptor peptide binding molecule binds specifically to anaffinity molecule. In some embodiments, the affinity molecule isstreptavidin, NeutrAvidin, or a derivative thereof. In some embodiments,the affinity acceptor peptide binding molecule does not specificallyinteract with an amino acid sequence of the encoded recombinant class Ior class II HLA. In some embodiments, for both the first and the secondrecombinant polynucleic acids: the sequence encoding an affinityacceptor tagged HLA is stably integrated into the genome of a cell. Insome embodiments, the sequence encoding β2 microglobulin or the sequenceencoding a HLA class II β-chain is connected to a sequence encoding asecond affinity acceptor peptide.

In some embodiments, the second affinity acceptor peptide comprises anHA tag. In some embodiments, the second affinity acceptor peptide cancomprise a biotin acceptor peptide (BAP), poly-histidine tag,poly-histidine-glycine tag, poly-arginine tag, poly-aspartate tag,poly-cysteine tag, poly-phenylalanine, c-myc tag, Herpes simplex virusglycoprotein D (gD) tag, FLAG tag, KT3 epitope tag, tubulin epitope tag,T7 gene 10 protein peptide tag, streptavidin tag, streptavidin bindingpeptide (SPB) tag, Strep-tag, Strep-tag II, albumin-binding protein(ABP) tag, alkaline phosphatase (AP) tag, bluetongue virus tag (B-tag),calmodulin binding peptide (CBP) tag, chloramphenicol acetyl transferase(CAT) tag, choline-binding domain (CBD) tag, chitin binding domain (CBD)tag, cellulose binding domain (CBP) tag, dihydrofolate reductase (DHFR)tag, galactose-binding protein (GBP) tag, maltose binding protein (MBP),glutathione-S-transferase (GST), Glu-Glu (EE) tag, human influenzahemagglutinin (HA) tag, horseradish peroxidase (HRP) tag, NE-tag, HSVtag, ketosteroid isomerase (KSI) tag, KT3 tag, LacZ tag, luciferase tag,NusA tag, PDZ domain tag, AviTag, Calmodulin-tag, E-tag, S-tag, SBP-tag,Softag 1, Softag 3, TC tag, VSV-tag, Xpress tag, Isopeptag, SpyTag,SnoopTag, Profinity eXact tag, Protein C tag, S1-tag, S-tag,biotin-carboxy carrier protein (BCCP) tag, green fluorescent protein(GFP) tag, small ubiquitin-like modifier (SUMO) tag, tandem affinitypurification (TAP) tag, HaloTag, Nus-tag, Thioredoxin-tag, Fc-tag, CYDtag, HPC tag, TrpE tag, ubiquitin tag, VSV-G epitope tag, V5 tag, or acombination thereof; optionally, wherein the second affinity acceptorpeptide comprises two or more repeats of a tag sequence.

In some embodiments, for both the first and the second recombinantpolynucleic acids: the sequence encoding β2 microglobulin or thesequence encoding a HLA class II β-chain is connected to the sequenceencoding a different recombinant HLA and the affinity acceptor peptideby a linker. In some embodiments, the linker comprises a polynucleicacid sequence encoding a cleavable linker. In some embodiments, thecleavable linker is a ribosomal skipping site or an internal ribosomalentry site (IRES) element. In some embodiments, the ribosomal skippingsite or IRES is cleaved when expressed in the cells. In someembodiments, the ribosomal skipping site is selected from the groupconsisting of F2A, T2A, P2A, and E2A. In some embodiments, the IRESelement is selected from common cellular or viral IRES sequences.

Provided herein is a composition comprising a first and a secondisolated polypeptide molecule encoded by the first and the secondpolynucleic acids, respectively of a composition described herein.Provided herein is a composition comprising a first and a second cellcomprising a first and a second polypeptide molecule encoded by thefirst and the second polynucleic acids, respectively of a compositiondescribed herein. Provided herein is a composition comprising a firstand a second cell comprising the first and the second polynucleic acids,respectively of a composition described herein. Provided herein is acomposition comprising a first and a second population of cellscomprising one or more cells comprising the first and the secondpolynucleic acids, respectively of a composition described herein.

In some embodiments, the first and the second population of cellsexpress one or more endogenous class I or class II HLA alleles. In someembodiments, the first and the second population of cells are engineeredto lack one or more endogenous HLA class I alleles. In some embodiments,the first and the second population of cells are engineered to lackendogenous HLA class I alleles. In some embodiments, the first and thesecond population of cells are engineered to lack one or more endogenousHLA class II alleles. In some embodiments, the first and the secondpopulation of cells are engineered to lack endogenous HLA class IIalleles. In some embodiments, the first and the second population ofcells are engineered to lack endogenous HLA class I alleles andendogenous HLA class II alleles.

Provided herein is a method of making a cell comprising transducing ortransfecting a first and a second cell with the first and the secondpolynucleic acids, respectively of a composition described herein.

Provided herein is a peptide identified according to a method describedherein.

Provided herein is a method of enriching for immunogenic peptidescomprising: providing a population of cells comprising one or more cellsexpressing an affinity acceptor tagged HLA, wherein the affinityacceptor tagged HLA comprises an affinity acceptor peptide operativelylinked to a recombinant HLA encoded by a recombinant HLA allele; andenriching for HLA-peptide complexes comprising the affinity acceptortagged HLA. In some embodiments, the method further comprisesdetermining the sequence of immunogenic peptides isolated from theHLA-peptide complexes. In some embodiments, the determining comprisesusing LC-MS/MS.

Human Leukocyte Antigen (HLA) System

The immune system can be classified into two functional subsystems: theinnate and the adaptive immune system. The innate immune system is thefirst line of defense against infections, and most potential pathogensare rapidly neutralized by this system before they can cause, forexample, a noticeable infection. The adaptive immune system reacts tomolecular structures, referred to as antigens, of the intrudingorganism. Unlike the innate immune system, the adaptive immune system ishighly specific to a pathogen. Adaptive immunity can also providelong-lasting protection; for example, someone who recovers from measlesis now protected against measles for their lifetime. There are two typesof adaptive immune reactions, which include the humoral immune reactionand the cell-mediated immune reaction. In the humoral immune reaction,antibodies secreted by B cells into bodily fluids bind topathogen-derived antigens, leading to the elimination of the pathogenthrough a variety of mechanisms, e.g. complement-mediated lysis. In thecell-mediated immune reaction, T-cells capable of destroying other cellsare activated. For example, if proteins associated with a disease arepresent in a cell, they are fragmented proteolytically to peptideswithin the cell. Specific cell proteins then attach themselves to theantigen or peptide formed in this manner and transport them to thesurface of the cell, where they are presented to the molecular defensemechanisms, in T cells, of the body. Cytotoxic T cells recognize theseantigens and kill the cells that harbor the antigens.

The term “major histocompatibility complex (MHC)”, “MHC molecules”, or“MHC proteins” refers to proteins capable of binding peptides resultingfrom the proteolytic cleavage of protein antigens and representingpotential T-cell epitopes, transporting them to the cell surface andpresenting them there to specific cells, e.g., in cytotoxicT-lymphocytes or T-helper cells. The human MHC is also called the HLAcomplex. Thus, the term “human leukocyte antigen (HLA) system”, “HLAmolecules” or “HLA proteins” refers to a gene complex encoding the MHCproteins in humans. The term MHC is referred as the “H-2” complex inmurine species. Those of ordinary skill in the art will recognize thatthe terms “major histocompatibility complex (MHC)”, “MHC molecules”,“MEW proteins” and “human leukocyte antigen (HLA) system”, “HLAmolecules”, “HLA proteins” are used interchangeably herein.

HLA proteins are classified into two types, referred to as HLA class Iand HLA class II. The structures of the proteins of the two HLA classesare very similar; however, they have very different functions. Class IHLA proteins are present on the surface of almost all cells of the body,including most tumor cells. Class I HLA proteins are loaded withantigens that usually originate from endogenous proteins or frompathogens present inside cells, and are then presented to naïve orcytotoxic T-lymphocytes (CTLs). HLA class II proteins are present onantigen presenting cells (APCs), including but not limited to dendriticcells, B cells, and macrophages. They mainly present peptides, which areprocessed from external antigen sources, i.e. outside of the cells, tohelper T cells. Most of the peptides bound by the HLA class I proteinsoriginate from cytoplasmic proteins produced in the healthy host cellsof an organism itself, and do not normally stimulate an immune reaction.

Class I HLA molecules consist of a heavy chain and a light chain and arecapable of binding a peptide of about 7 to 13 amino acids (e.g., about 8to 11 amino acids, or 9 or 10 amino acids), if this peptide has suitablebinding motifs, and presenting it to cytotoxic T-lymphocytes. Thepeptides bound by class I HLA molecules originate from an endogenousprotein antigen. The heavy chain of the HLA molecules of class I can bean HLA-A, HLA-B or HLA-C monomer, and the light chain isβ-2-microglobulin. Class I HLA occurs as an a chain composed of threedomains—α1, α2, and α3. This chain is often referred to as the class Iheavy chain, and is referred to herein as the class I alpha-chain. Theα1 rests upon a unit of the non-HLA molecule β2 microglobulin (encodedon human chromosome 15). The α3 domain is transmembrane, anchoring theHLA class I molecule to the cell membrane. The peptide being presentedis held by the floor of the peptide-binding groove, in the centralregion of the α1/α2 heterodimer (a molecule composed of two nonidenticalsubunits). Class I HLA-A, HLA-B or HLA-C are highly polymorphic. ClassIb HLA exhibits limited polymorphism, expression patterns and presentedantigens. This group is subdivided into a group encoded within HLA loci,e.g., HLA-E, HLA-F, HLA-G, as well as those not, e.g., stress ligandssuch as ULBPs, Rae1 and H60. The antigen/ligand for many of thesemolecules remain unknown, but they can interact with each of CD8+ Tcells, NKT cells, and NK cells.

In some embodiments, the present disclosure utilizes a non-classicalclass I HLA-E allele. HLA-E is one of non-classical class I moleculerecognized by natural killer (NK) cells and CD8⁺ T cells. HLA-E isexpressed in almost all tissues including lung, liver, skin andplacental cells. HLA-E expression is also detected in solid tumors(e.g., osteosarcoma and melanoma). HLA-E binds to TCR expressed on CD8⁺T cells, resulting in the T cell activation. HLA-E is also known to bindCD94/NKG2 receptor expressed on NK cells and CD8⁺ T cells. CD94 can pairwith several different isoforms of NKG2 to form receptors with potentialto either inhibit (NKG2A, NKG2B) or promote (NKG2C) cellular activation.HLA-E can bind to a peptide derived from amino acid residues 3-11 of theleader sequences of most HLA-A, -B, -C, and -G molecules, but cannotbind its own leader peptide. HLA-E has also been shown to presentpeptides derived from endogenous proteins similar to HLA-A, -B, and -Calleles. Under physiological conditions, the engagement of CD94/NKG2Awith HLA-E, loaded with peptides from the HLA class I leader sequences,usually induces inhibitory signals. Cytomegalovirus (CMV) utilizes themechanism for escape from NK cell immune surveillance via expression ofthe UL40 glycoprotein, mimicking the HLA-A leader. However, it is alsoreported that CD8⁺ T cells can recognize HLA-E loaded with the UL40peptide derived from CMV Toledo strain and play a role in defenseagainst CMV. A number of studies revealed several important functions ofHLA-E in infectious disease and cancer.

The peptide antigens attach themselves to the molecules of HLA class Iby competitive affinity binding within the endoplasmic reticulum, beforethey are presented on the cell surface. Here, the affinity of anindividual peptide antigen is directly linked to its amino acid sequenceand the presence of specific binding motifs in defined positions withinthe amino acid sequence. If the sequence of such a peptide is known, itis possible to manipulate the immune system against diseased cellsusing, for example, peptide vaccines.

Class II HLA molecules have two chains, α and β, each having twodomains—α1 and α2 and β1 and β2—each chain having a transmembranedomain, α2 and β2, respectively, anchoring the HLA class II molecule tothe cell membrane. The peptide-binding groove is formed of theheterodimer of α1 and β1. The peptide bound by the HLA molecules ofclass II usually originates from an extracellular of exogenous proteinantigen. The α-chain and the β-chain are in HLA-DR, HLA-DQ and HLA-DPmonomers (FIG. 1B). Class II HLA molecules have six isotypes. Classicmolecules present peptides to CD4+ lymphocytes. Nonclassic molecules,accessories, with intracellular functions, are not exposed on cellmembranes, but in internal membranes in lysosomes, normally loading theantigenic peptides onto classic HLA class II molecules.

In HLA class II, phagocytes such as macrophages and immature dendriticcells take up entities by phagocytosis into phagosomes—though B cellsexhibit the more general endocytosis into endosomes—which fuse withlysosomes whose acidic enzymes cleave the uptaken protein into manydifferent peptides. Authophagy is another source of HLA class IIpeptides. Via physicochemical dynamics in molecular interaction with theHLA class II variants borne by the host, encoded in the host's genome, aparticular peptide exhibits immunodominance and loads onto HLA class IImolecules. These are trafficked to and externalized on the cell surface.The most studied subclass II HLA genes are: HLA-DPA1, HLA-DPB1,HLA-DQA1, HLA-DQB1, HLA-DRA, and HLA-DRB1.

Presentation of peptides by HLA class II molecules to CD4+ helper Tcells is required for immune responses to foreign antigens (Roche andFuruta, 2015). Once activated, CD4+ T cells promote B celldifferentiation and antibody production, as well as CD8+ T cell (CTL)responses. CD4+ T cells also secrete cytokines and chemokines thatactivate and induce differentiation of other immune cells. HLA class IImolecules are heterodimers of α and β chains that interact to form apeptide-binding groove that is more open than class I peptide-bindinggrooves (Unanue et al., 2016). Peptides bound to HLA class II moleculesare believed to have a 9-amino acid binding core with flanking residueson either N- or C-terminal side that overhang from the groove (Jardetzkyet al., 1996; Stern et al., 1994). These peptides are usually 12-16amino acids in length and often contain 3-4 anchor residues at positionsP1, P4, P6/7 and P9 of the binding register (Rossjohn et al., 2015).

HLA alleles are expressed in codominant fashion, meaning that thealleles (variants) inherited from both parents are expressed equally.For example, each person carries 2 alleles of each of the 3 class Igenes, (HLA-A, HLA-B and HLA-C), and so can express six different typesof class II HLA. In the class II HLA locus, each person inherits a pairof HLA-DP genes (DPA1 and DPB1, which encode α and β chains), a coupleof genes HLA-DQ (DQA1 and DQB1, for α and β chains), one gene HLA-DRα(DRA1), and one or more genes HLA-DRβ (DRB1 and DRB3, -4 or -5). Thatmeans that one heterozygous individual can inherit six or eightfunctioning class II HLA alleles, three or more from each parent. Thus,the HLA genes are highly polymorphic; many different alleles exist inthe different individuals inside a population. Genes encoding HLAproteins have many possible variations, allowing each person's immunesystem to react to a wide range of foreign invaders. Some HLA genes havehundreds of identified versions (alleles), each of which is given aparticular number. In some embodiments, the class I HLA alleles areHLA-A*02:01, HLA-B*14:02, HLA-A*23:01, HLA-E*01:01 (non-classical). Insome embodiments, class II HLA alleles are HLA-DRB*01:01, HLA-DRB*01:02,HLA-DRB*11:01, HLA-DRB*15:01, and HLA-DRB*07:01.

Subject specific HLA alleles or HLA genotype of a subject can bedetermined by any method known in the art. In exemplary embodiments, HLAgenotypes are determined by any method described in International PatentApplication number PCT/US2014/068746, published Jun. 11, 2015 asWO2015085147. Briefly, the methods include determining polymorphic genetypes that can comprise generating an alignment of reads extracted froma sequencing data set to a gene reference set comprising allele variantsof the polymorphic gene, determining a first posterior probability or aposterior probability derived score for each allele variant in thealignment, identifying the allele variant with a maximum first posteriorprobability or posterior probability derived score as a first allelevariant, identifying one or more overlapping reads that aligned with thefirst allele variant and one or more other allele variants, determininga second posterior probability or posterior probability derived scorefor the one or more other allele variants using a weighting factor,identifying a second allele variant by selecting the allele variant witha maximum second posterior probability or posterior probability derivedscore, the first and second allele variant defining the gene type forthe polymorphic gene, and providing an output of the first and secondallele variant.

As described herein, there is a large body of evidence in both animalsand humans that mutated epitopes are effective in inducing an immuneresponse and that cases of spontaneous tumor regression or long termsurvival correlate with CD8+ T-cell responses to mutated epitopes(Buckwalter and Srivastava P K. “It is the antigen(s), stupid” and otherlessons from over a decade of vaccitherapy of human cancer. Seminars inimmunology 20:296-300 (2008); Karanikas et al, High frequency ofcytolytic T lymphocytes directed against a tumor-specific mutatedantigen detectable with HLA tetramers in the blood of a lung carcinomapatient with long survival. Cancer Res. 61:3718-3724 (2001); Lennerz etal. The response of autologous T cells to a human melanoma is dominatedby mutated neoantigens. Proc Natl Acad Sci USA. 102:16013 (2005)) andthat “immunoediting” can be tracked to alterations in expression ofdominant mutated antigens in mice and man (Matsushita et al, Cancerexome analysis reveals a T-cell-dependent mechanism of cancerimmunoediting Nature 482:400 (2012); DuPage et al, Expression oftumor-specific antigens underlies cancer immunoediting Nature 482:405(2012); and Sampson et al, Immunologic escape after prolongedprogression-free survival with epidermal growth factor receptor variantIII peptide vaccination in patients with newly diagnosed glioblastoma JClin Oncol. 28:4722-4729 (2010)).

Sequencing technology has revealed that each tumor contains multiple,patient-specific mutations that alter the protein coding content of agene. Such mutations create altered proteins, ranging from single aminoacid changes (caused by missense mutations) to addition of long regionsof novel amino acid sequence due to frame shifts, read-through oftermination codons or translation of intron regions (novel open readingframe mutations; neoORFs). These mutated proteins are valuable targetsfor the host's immune response to the tumor as, unlike native proteins,they are not subject to the immune-dampening effects of self-tolerance.Therefore, mutated proteins are more likely to be immunogenic and arealso more specific for the tumor cells compared to normal cells of thepatient.

The term “T cell” includes CD4+ T cells and CD8+ T cells. The term Tcell also includes both T helper 1 type T cells and T helper 2 type Tcells. T cells as used herein are generally classified by function andcell surface antigens (cluster differentiation antigens, or CDs), whichalso facilitate T cell receptor binding to antigen, into two majorclasses: helper T (T_(H)) cells and cytotoxic T-lymphocytes (CTLs).

Mature helper T (T_(H)) cells express the surface protein CD4 and arereferred as CD4+ T cells. Following T cell development, matured, naïve Tcells leave the thymus and begin to spread throughout the body,including the lymph nodes. Naïve T cells are those T cells that havenever been exposed to the antigen that they are programmed to respondto. Like all T cells, they express the T cell receptor-CD3 complex. TheT cell receptor (TCR) consists of both constant and variable regions.The variable region determines what antigen the T cell can respond to.CD4+ T cells have TCRs with an affinity for Class II MHC, and CD4 isinvolved in determining MHC affinity during maturation in the thymus.Class II MHC proteins are generally only found on the surface ofspecialized antigen-presenting cells (APCs). Specialized antigenpresenting cells (APCs) are primarily dendritic cells, macrophages and Bcells, although dendritic cells are the only cell group that expressesMHC Class II constitutively (at all times). Some APCs also bind native(or unprocessed) antigens to their surface, such as follicular dendriticcells, but unprocessed antigens do not interact with T cells and are notinvolved in their activation. The peptide antigens that bind to MEWclass I proteins are typically shorter than peptide antigens that bindto MEW class II proteins.

Cytotoxic T-lymphocytes (CTLs), also known as cytotoxic T cells,cytolytic T cells, CD8+ T cells, or killer T cells, refer to lymphocyteswhich induce apoptosis in targeted cells. CTLs form antigen-specificconjugates with target cells via interaction of TCRs with processedantigen (Ag) on target cell surfaces, resulting in apoptosis of thetargeted cell. Apoptotic bodies are eliminated by macrophages. The term“CTL response” is used to refer to the primary immune response mediatedby CTL cells. Cytotoxic T-lymphocytes have both T-cell receptors (TCR)and CD8 molecules on their surface. T cell receptors are capable ofrecognizing and binding peptides complexed with the molecules of HLAclass I. Each cytotoxic T-lymphocyte expresses a unique T-cell receptorwhich is capable of binding specific MHC/peptide complexes. Mostcytotoxic T cells express T-cell receptors (TCRs) that can recognize aspecific antigen. In order for the TCR to bind to the class I MEWmolecule, the former must be accompanied by a glycoprotein called CD8,which binds to the constant portion of the class I MHC molecule.Therefore, these T cells are called CD8+ T cells. The affinity betweenCD8 and the MHC molecule keeps the T cell and the target cell boundclosely together during antigen-specific activation. CD8+ T cells arerecognized as T cells once they become activated and are generallyclassified as having a pre-defined cytotoxic role within the immunesystem. However, CD8+ T cells also have the ability to make somecytokines.

“T cell receptors (TCR)” are cell surface receptors that participate inthe activation of T cells in response to the presentation of antigen.The TCR is generally made from two chains, alpha and beta, whichassemble to form a heterodimer and associates with the CD3-transducingsubunits to form the T-cell receptor complex present on the cellsurface. Each alpha and beta chain of the TCR consists of animmunoglobulin-like N-terminal variable (V) and constant (C) region, ahydrophobic transmembrane domain, and a short cytoplasmic region. As forimmunoglobulin molecules, the variable regions of the alpha and betachains are generated by V(D)J recombination, creating a large diversityof antigen specificities within the population of T cells. However, incontrast to immunoglobulins that recognize intact antigen, T cells areactivated by processed peptide fragments in association with an MHCmolecule, introducing an extra dimension to antigen recognition by Tcells, known as MHC restriction. Recognition of MHC disparities betweenthe donor and recipient through the T cell receptor leads to T cellproliferation and the potential development of GVHD. It has been shownthat normal surface expression of the TCR depends on the coordinatedsynthesis and assembly of all seven components of the complex (Ashwelland Klusner 1990). The inactivation of TCRα or TCRβ can result in theelimination of the TCR from the surface of T cells preventingrecognition of alloantigen and thus GVHD. However, TCR disruptiongenerally results in the elimination of the CD3 signaling component andalters the means of further T cell expansion.

The term “HLA peptidome” refers to a pool of peptides which specificallyinteracts with a particular HLA class and can encompass thousands ofdifferent sequences. HLA peptidomes include a diversity of peptides,derived from both normal and abnormal proteins expressed in the cells.Thus, the HLA peptidomes can be studied to identify cancer specificpeptides, for development of tumor immunotherapeutics and as a source ofinformation about protein synthesis and degradation schemes within thecancer cells. In some embodiments, HLA peptidome is a pool of solubleHLA molecules (sHLA). In some embodiments, HLA peptidome is a pool ofmembranal HLA (mHLA).

The term “antigen presenting cell” or “APC” includes professionalantigen presenting cells (e.g., B lymphocytes, macrophages, monocytes,dendritic cells, Langerhans cells), as well as other antigen presentingcells (e.g., keratinocytes, endothelial cells, astrocytes, fibroblasts,oligodendrocytes, thymic epithelial cells, thyroid epithelial cells,glial cells (brain), pancreatic beta cells, and vascular endothelialcells). An “antigen presenting cell” or “APC” is a cell that expressesthe Major Histocompatibility complex (MHC) molecules and can displayforeign antigen complexed with MHC on its surface.

Universal IP Pipeline: Universal Mono-Allelic HLA-Peptide ComplexIdentification Platform

Adaptive immune responses rely, in part, on the ability of cytotoxicCD8⁺ T cells to identify and eliminate cells that displaydisease-associated antigens bound to human leukocyte antigen (HLA) classI molecules. HLA class I proteins (HLA-A, B and C) are expressed on thesurface of almost all nucleated cells in the human body and are requiredfor presentation of short peptides for detection by CD8⁺ T cellreceptors. The HLA-bound peptides arise from endogenous or foreignproteins that are cleaved by the proteasome and ER peptidases prior toloading and display by HLA class I proteins. The HLA genes are the mostpolymorphic genes across the human population, with more than 10,000 HLAclass I allele variants identified to date (Robinson et al., 2015). EachHLA allele is estimated to bind and present ˜1,000-10,000 uniquepeptides to T cells; ≤0.1% of ˜10 million potential 9mer peptides fromhuman protein-coding genes (Bassani-Sternberg et al., 2015; Hunt et al.,1992; Rammensee et al., 1995, 1999; Rock et al.; Vita et al., 2015; Walzet al., 2015).

Unlike class I, HLA class II proteins (HLA-DR, DQ and DP) are onlyexpressed on the surface of antigen presenting cells (APCs) andepithelial, vascular and connective tissues cells in response toinflammatory signals. Presentation of peptides, most often derived fromexogenous proteins, by HLA class II molecules to CD4+ T cells isrequired for immune responses to foreign antigens (Roche and Furuta,2015). Once activated, CD4+ T cells promote B cell differentiation andantibody production, as well as CD8+ T cell responses. CD4+ T cells alsosecrete cytokines and chemokines that activate and inducedifferentiation of other immune cells. HLA class II molecules areheterodimers of α and β chains that interact to form a peptide-bindinggroove that is more open than class I peptide-binding grooves (Unanue etal., 2016). Peptides bound to HLA class II molecules are believed tohave a 9-amino acid binding core with flanking residues on either N- orC-terminal side that overhang from the groove (Jardetzky et al., 1996;Stern et al., 1994). These peptides are usually 12-16 amino acids inlength and often contain 3-4 anchor residues at positions P1, P4, P6/7and P9 of the binding register (Rossjohn et al., 2015). Less is knownabout allele-specific peptide-binding characteristics of HLA class IImolecules because of the heterogeneity of α and β chain pairing,complexity of data limiting the ability to confidently assign corebinding epitopes, and the lack of immunoprecipitation grade,allele-specific antibodies required for high-resolution biochemicalanalyses.

Peptide-binding rules have been studied extensively for a subset of HLAalleles (Vita et al., 2015) and encoded in advanced neural network-basedalgorithms that predict binding (Hoof et al., 2009; Lundegaard et al.,2008). However, several factors limit the power to predict peptidespresented on HLA alleles. First, the provenance of peptide data uponwhich these algorithms are trained is diverse, ranging from peptidelibrary screens to Edman degradation and mass spectrometry-basedsequencing of endogenously processed and presented peptides (Boen etal., 2000; Rammensee et al., 1995, 1999; Vita et al., 2015). Massspectrometry-based peptide identifications make up around 30% of thetotal identification in IEDB. Mass spectrometry has become a desiredmethod of HLA-associated peptide sequencing because of pioneering workby Donald F. Hunt and colleagues (Cobbold et al., 2013; Hunt et al.,1992; Meadows et al., 1997; Mohammed et al., 2008; Zarling et al., 2000,2006), as well as improvements to instrumentation demonstrated by manygroups over the past two decades (Bassani-Sternberg et al., 2015; Caronet al., 2015; Mommen et al., 2014). Second, many existing predictionalgorithms have focused on predicting binding but may not fully takeinto account endogenous processes that generate and transport peptidesprior to binding (Larsen et al., 2007). Third, the number of bindingpeptides for many HLA alleles is too small to develop a reliablepredictor. Until now, however, the generation of high-quality resourcedatasets has been hampered by inefficient protocols that necessitateprohibitively large amounts of input cellular material and a lack ofdatabase search tools for HLA-peptide sequencing (Caron et al., 2015;Hoof et al., 2009; Lundegaard et al., 2008; Vita et al., 2015).

Disclosed herein is a unique biochemical enrichment strategy forpeptide-HLA class I and II complexes from live cells and cellularlysate. HLA molecules containing an N-terminal or C-terminal tagsequence (e.g., BAP or HA) can be labeled on the cell surface or in celllysate. For example, HLA molecules containing an N-terminal orC-terminal biotin acceptor peptide (BAP) sequence can be enzymaticallylabeled with biotin on the cell surface or in cell lysate. For example,HLA molecules containing an N-terminal or C-terminal HA sequence can beenriched from complex cellular mixtures using an HA-specific antibody.In an exemplary embodiment, biotin labeled HLA-peptide complexes areenriched from complex cellular mixtures using streptavidin/NeutrAvidinbeads and the enriched HLA-peptide complexes are analyzed orcharacterized. In an exemplary embodiment, HA labeled HLA-peptidecomplexes are enriched from complex cellular mixtures using anHA-specific antibody and the enriched HLA-peptide complexes are analyzedor characterized. For example, associated peptides can be eluted andsequenced by LC-MS/MS. Importantly, the presently disclosed methodsprovide a universal platform for analyzing and characterizingHLA-peptide complexes. For example, the presently disclosed methodsprovide a universal platform for the identification of endogenouslypresented peptides from cell line expressing all possible class I or IIconstructs.

Disclosed herein are single HLA class I and class II allele-expressingcell lines enabling unambiguous peptide:allele assignments (Shimizu andDeMars, 1989; Shimizu et al., 1986). This is an improvement upon currentHLA-bound peptide detection methods as most MS-based studies involveeluting and sequencing a messy admixture of ligands bound to multipleHLA-A,B, and C molecules, which require affinity predictions andsometimes deconvolution for allele assignments (Bassani-Sternberg andGfeller, 2016). Studies with soluble HLA transfected cell lines havebeen able to derive peptide-binding epitopes for a single HLA allele,but the most comprehensive experiments to date have identified only <200unique peptides and have required several orders of magnitude morestarting cellular material (Hawkins et al., 2008). By removing theuncertainty of peptide:HLA assignments, presently disclosed methodsfacilitate deeper and more precise evaluations of HLA-peptide ligandomesand rules related to peptide antigen processing and presenting usingless cellular material than previous efforts.

The methods and compositions described herein include, for example,chemically labeled variable β-chain (biotinylation) to differentiatebetween class II HLA heterodimers presented by cells, which allow forimproved epitope mapping. HLA class I and class II constructs thatcontain a tag, such as a biotin acceptor peptide sequence (BAP), at theN- or C-terminus, can be used in the methods described herein. N- andC-terminal affinity tagging enables HLA-allele selectiveimmunopurification from cells expressing endogenous HLA. N-terminalaffinity tagging enables HLA-allele selective immunopurification ofcomplexes presented on the cell surface. For example, after transfectionor transduction, N-terminal biotinylation enables differentiationbetween HLA complexes presented on the cell surface vs. all HLA-peptidecomplexes in cellular lysates. For example, biotinylation of HLA-peptidecomplexes on intact cell surfaces (no lysis) enables unbiased massspectrometry (MS) sequencing methods of endogenously processed andpresented peptides. The enrichment methods disclosed herein, such asimmunoprecipitation enrichment methods, enable high throughput analysisof cell samples.

Provided herein is a method of characterizing HLA-peptide complexescomprising: providing a population of cells, wherein one or more cellsof the population of cells comprise a polynucleic acid comprising asequence encoding an affinity acceptor tagged class I or class II HLAallele, wherein the sequence encoding an affinity acceptor tagged HLAcomprises a sequence encoding a recombinant class I or class II HLAallele operatively linked to a sequence encoding an affinity acceptorpeptide; expressing the affinity acceptor tagged HLA in at least onecell of the one or more cells of the population of cells, therebyforming affinity acceptor tagged HLA-peptide complexes in the at leastone cell; enriching for the affinity acceptor tagged HLA-peptidecomplexes; and characterizing HLA-peptide complexes.

In some embodiments, the characterizing comprises characterizing apeptide bound to the affinity acceptor tagged HLA-peptide complex fromthe enriching.

In some embodiments, the method comprises carrying out the steps of themethod for two or more class I and/or class II HLA alleles. In someembodiments, the two or more class I and/or class II HLA allelescomprise at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 25, 30, 35, 40, 45, or 50 class I and/or class II HLAalleles.

In some embodiments, the affinity acceptor tagged HLA-peptide complexescomprise a transmembrane domain. In some embodiments, the affinityacceptor tagged HLA-peptide complexes comprise an intracellular domain.In some embodiments, the affinity acceptor tagged HLA-peptide complexesare not excreted. In some embodiments, the affinity acceptor taggedHLA-peptide complexes incorporate into a cell membrane when expressed.In some embodiments, the affinity acceptor tagged HLA-peptide complexesare not soluble affinity acceptor tagged HLA-peptide complexes.

In some embodiments, the method further comprises generating anHLA-allele specific peptide database.

In some embodiments, the recombinant class I or class II HLA allele is asingle recombinant class I or class II HLA allele.

In some embodiments, method comprises: providing a population of cellseach comprising one or more cells comprising an affinity acceptor taggedHLA, wherein the affinity acceptor tagged HLA comprises a differentrecombinant polypeptide encoded by a different HLA allele operativelylinked to an affinity acceptor peptide; enriching for affinity acceptortagged HLA-peptide complexes; and characterizing a peptide or a portionthereof bound to the affinity acceptor tagged HLA-peptide complex fromthe enriching.

In some embodiments, the method comprises introducing one or morepeptides to the population of cells.

In some embodiments, the introducing comprises contacting the populationof cells with the one or more peptides or expressing the one or morepeptides in the population of cells. In some embodiments, theintroducing comprises contacting the population of cells with one ormore nucleic acids encoding the one or more peptides. In someembodiments, the one or more nucleic acids encoding the one or morepeptides is DNA. In some embodiments, the one or more nucleic acidsencoding the one or more peptides is RNA, optionally wherein the RNA ismRNA.

In some embodiments, the enriching does not comprise use of a tetramerreagent.

In some embodiments, the characterizing comprises determining thesequence of a peptide or a portion thereof bound to the affinityacceptor tagged HLA-peptide complex from the enriching, optionallydetermining whether a peptide or a portion thereof is modified. In someembodiments, the determining comprises biochemical analysis, massspectrometry analysis, MS analysis, MS/MS analysis, LC-MS/MS analysis,or a combination thereof. In some embodiments, the characterizingcomprises evaluating a binding affinity or stability of a peptide or aportion thereof bound to the affinity acceptor tagged HLA-peptidecomplex from the enriching. In some embodiments, the characterizingcomprises determining whether a peptide or a portion thereof bound tothe affinity acceptor tagged HLA-peptide complex from the enrichingcontains one or more mutations. In some embodiments, the characterizingcomprises evaluating associations of peptides with HLA molecules in theaffinity acceptor tagged HLA-peptide complexes.

In some embodiments, the method comprises expressing a library ofpeptides in the population of cells, thereby forming a library ofaffinity acceptor tagged HLA-peptide complexes. In some embodiments, themethod comprises contacting to the population of cells a library ofpeptides or a library of sequences encoding peptides, thereby forming alibrary of affinity acceptor tagged HLA-peptide complexes. In someembodiments, the library comprises a library of peptides associated witha disease or condition. In some embodiments, the disease or condition iscancer, an infection with an infectious agent, or an autoimmunereaction. In some embodiments, the method comprises introducing theinfectious agent or portions thereof into one or more cells of thepopulation of cells. In some embodiments, the method comprisescharacterizing one or more peptides from the HLA-peptide complexes,optionally wherein the peptides are from one or more target proteins ofthe infectious agent. In some embodiments, the method comprisescharacterizing one or more regions of the peptides from the one or moretarget proteins of the infectious agent. In some embodiments, the methodcomprises identifying peptides from the HLA-peptide complexes derivedfrom an infectious agent.

In some embodiments, the population of cells is from a biological samplefrom a subject with a disease or condition. In some embodiments, thepopulation of cells is a cell line. In some embodiments, the populationof cells is a population of primary cells. In some embodiments, therecombinant class I or class II HLA allele is matched to a subject witha disease or condition.

In some embodiments, the method comprises screening for drug (e.g.,biologics) hypersensitivity. In some embodiments, the method comprisesassessing whether an administered biologic (e.g., a protein, peptide orantibody drug), a fragment of administered biologics, or a processedbiologic fragment are presented to T cells. These epitopes can causeadverse effect in the subject, and thus how administered biologics areprocessed in the subject should be monitored. For example, an HIV drug(e.g., Abacavir) can bind to HLA molecules and change peptide-bindingmotifs for certain HLA alleles (e.g., HLA-B5701).

In some embodiments, the peptide from the affinity acceptor taggedHLA-peptide complex is capable of activating a T cell from a subjectwhen presented by an antigen presenting cell. In some embodiments, thecharacterizing comprises comparing HLA-peptide complexes from cancercells to HLA-peptide complexes from non-cancer cells.

In some embodiments, the population of cells comprises a plurality ofpopulations of cells, each population of cells expressing a differentrecombinant class I or class II HLA allele. In some embodiments, eachpopulation of cells of the plurality is in a same or a separatecontainer.

In some embodiments, the method further comprises isolating peptidesfrom the affinity acceptor tagged HLA-peptide complexes before thecharacterizing. In some embodiments, the method further comprisesremoving one or more amino acids from a terminus of a peptide bound toan affinity acceptor tagged HLA-peptide complex.

In some embodiments, the population of cells is a population of low cellsurface HLA class I or class II expressing cells. In some embodiments,the population of cells expresses one or more endogenous HLA alleles. Insome embodiments, the population of cells is an engineered population ofcells lacking one or more endogenous HLA class I alleles. In someembodiments, the population of cells is an engineered population ofcells lacking endogenous HLA class I alleles. In some embodiments, thepopulation of cells is an engineered population of cells lacking one ormore endogenous HLA class II alleles. In some embodiments, thepopulation of cells is an engineered population of cells lackingendogenous HLA class II alleles. In some embodiments, the population ofcells is an engineered population of cells lacking endogenous HLA classI alleles and endogenous HLA class II alleles. In some embodiments, thepopulation of cells is a knock-out of one or more HLA class I alleles.

In some embodiments, the population of cells is a knock-out of one ormore HLA class II alleles. In some embodiments, the population of cellsis a knock-out of all HLA class I alleles. In some embodiments, thepopulation of cells is a knock-out of all HLA class II alleles In someembodiments, the population of cells is a knock-out of all HLA class Ialleles and a knock-out of all HLA class II alleles. In someembodiments, the sequence encoding the recombinant class I or class IIHLA allele encodes a class I HLA. In some embodiments, the class I HLAis selected from the group consisting of HLA-A, HLA-B, HLA-C, HLA-E,HLA-F, and HLA-G. In some embodiments, the sequence encoding therecombinant class I or class II HLA allele encodes a class II HLA. Insome embodiments, the class II HLA is selected from the group consistingof HLA-DR, HLA-DQ, and HLA-DP. In some embodiments, the class II HLAcomprises a HLA class II α-chain, a HLA class II β-chain, or acombination thereof.

In some embodiments, each sequence encodes at least two different classI and/or class II HLA alleles. In some embodiments, the at least twodifferent class I and/or class II HLA alleles are each operativelylinked to a sequence encoding a different affinity acceptor peptide. Insome embodiments, the at least two different class I and/or class II HLAalleles are each operatively linked to a sequence encoding the affinityacceptor peptide.

In some embodiments, the method comprises administering at least asecond polynucleic acid comprising a sequence encoding a differentrecombinant HLA allele operatively linked to the same or a differentaffinity acceptor peptide.

In some embodiments, the sequence encoding the affinity acceptor peptideis operatively linked to a sequence that encodes an extracellularportion of the recombinant class I or class II HLA allele.

In some embodiments, the encoded affinity acceptor peptide is expressedextracellularly. In some embodiments, the sequence encoding the affinityacceptor peptide is operatively linked to the N-terminus of the sequenceencoding the recombinant class I or class II HLA allele. In someembodiments, the sequence encoding the affinity acceptor peptide isoperatively linked to a sequence that encodes an intracellular portionof the recombinant class I or class II HLA allele. In some embodiments,the encoded affinity acceptor peptide is expressed intracellularly. Insome embodiments, the sequence encoding the affinity acceptor peptide isoperatively linked to the C-terminus of the sequence encoding therecombinant class I or class II HLA allele. In some embodiments, thesequence encoding the affinity acceptor peptide is operatively linked tothe sequence encoding the recombinant class I or class II HLA allele bya linker.

In some embodiments, enriching comprises enriching for intact cellsexpressing the affinity acceptor tagged HLA-peptide complexes. In someembodiments, the method does not comprise lysing the cells beforeenriching. In some embodiments, the method further comprises lysing theone or more cells before enriching. In some embodiments, enrichingcomprises contacting an affinity acceptor peptide binding molecule tothe affinity acceptor tagged HLA-peptide complexes, wherein the affinityacceptor peptide binding molecule binds specifically to the affinityacceptor peptide.

In some embodiments, the affinity acceptor peptide comprises a tagsequence comprising a biotin acceptor peptide (BAP), poly-histidine tag,poly-histidine-glycine tag, poly-arginine tag, poly-aspartate tag,poly-cysteine tag, poly-phenylalanine, c-myc tag, Herpes simplex virusglycoprotein D (gD) tag, FLAG tag, KT3 epitope tag, tubulin epitope tag,T7 gene 10 protein peptide tag, streptavidin tag, streptavidin bindingpeptide (SPB) tag, Strep-tag, Strep-tag II, albumin-binding protein(ABP) tag, alkaline phosphatase (AP) tag, bluetongue virus tag (B-tag),calmodulin binding peptide (CBP) tag, chloramphenicol acetyl transferase(CAT) tag, choline-binding domain (CBD) tag, chitin binding domain (CBD)tag, cellulose binding domain (CBP) tag, dihydrofolate reductase (DHFR)tag, galactose-binding protein (GBP) tag, maltose binding protein (MBP),glutathione-S-transferase (GST), Glu-Glu (EE) tag, human influenzahemagglutinin (HA) tag, horseradish peroxidase (HRP) tag, NE-tag, HSVtag, ketosteroid isomerase (KSI) tag, KT3 tag, LacZ tag, luciferase tag,NusA tag, PDZ domain tag, AviTag, Calmodulin-tag, E-tag, S-tag, SBP-tag,Softag 1, Softag 3, TC tag, VSV-tag, Xpress tag, Isopeptag, SpyTag,SnoopTag, Profinity eXact tag, Protein C tag, S1-tag, S-tag,biotin-carboxy carrier protein (BCCP) tag, green fluorescent protein(GFP) tag, small ubiquitin-like modifier (SUMO) tag, tandem affinitypurification (TAP) tag, HaloTag, Nus-tag, Thioredoxin-tag, Fc-tag, CYDtag, HPC tag, TrpE tag, ubiquitin tag, VSV-G epitope tag, V5 tag, or acombination thereof optionally, wherein the affinity acceptor peptidecomprises two or more repeats of a tag sequence. In some embodiments,the affinity acceptor peptide binding molecule is biotin or an antibodyspecific to the affinity acceptor peptide. In some embodiments, theenriching comprises contacting an affinity molecule to the affinityacceptor tagged HLA-peptide complexes, wherein the affinity moleculebinds specifically to the affinity acceptor peptide binding molecule. Insome embodiments, the affinity molecule is streptavidin, NeutrAvidin, ora derivative thereof. In some embodiments, enriching comprisesimmunoprecipitating affinity acceptor tagged HLA-peptide complexes. Insome embodiments, the affinity acceptor peptide binding molecule isattached to a solid surface. In some embodiments, the affinity moleculeis attached to a solid surface. In some embodiments, the solid surfaceis a bead. In some embodiments, enriching comprises immunoprecipitatingaffinity acceptor tagged HLA-peptide complexes with an affinity acceptorpeptide binding molecule that binds specifically to the affinityacceptor peptide. In some embodiments, the affinity acceptor peptidebinding molecule does not specifically interact with the amino acidsequence of the encoded recombinant class I or class II HLA. In someembodiments, enriching comprises contacting an affinity moleculespecific to an extracellular portion of the recombinant class I or classII HLA allele. In some embodiments, enriching comprises contacting anaffinity molecule specific to an N-terminal portion of the recombinantclass I or class II HLA allele.

In some embodiments, providing comprises contacting the population ofcells with the polynucleic acid. In some embodiments, contactingcomprises transfecting or transducing. In some embodiments, providingcomprises contacting the population of cells with a vector comprisingthe polynucleic acid. In some embodiments, the vector is a viral vector.In some embodiments, the polynucleic acid is stably integrated into thegenome of the population of cells.

In some embodiments, the sequence encoding the recombinant class I orclass II HLA comprises a sequence encoding a HLA class I α-chain. Insome embodiments, the method further comprises expressing a sequenceencoding β2 microglobulin in the one or more cells. In some embodiments,the sequence encoding β2 microglobulin is connected to the sequenceencoding the HLA class I α-chain. In some embodiments, the sequenceencoding β2 microglobulin is connected to the sequence encoding the HLAclass I α-chain by a linker. In some embodiments, the sequence encodingβ2 microglobulin is connected to a sequence encoding a second affinityacceptor peptide. In some embodiments, the sequence encoding therecombinant class I or class II HLA comprises a sequence encoding a HLAclass II α-chain. In some embodiments, the method further comprisesexpressing a sequence encoding a HLA class II β-chain in the one or morecells In some embodiments, the sequence encoding the HLA class IIβ-chain is connected to the sequence encoding the HLA class II α-chain.In some embodiments, the sequence encoding the HLA class II β-chain isconnected to the sequence encoding the HLA class II α-chain by a linker.

In some embodiments, the sequence encoding the HLA class II β-chain isconnected to a sequence encoding a second affinity acceptor peptide. Insome embodiments, the second affinity acceptor peptide is different thanthe first affinity acceptor peptide and is selected from the groupconsisting of biotin acceptor peptide (BAP), poly-histidine tag,poly-histidine-glycine tag, poly-arginine tag, poly-aspartate tag,poly-cysteine tag, poly-phenylalanine, c-myc tag, Herpes simplex virusglycoprotein D (gD) tag, FLAG tag, KT3 epitope tag, tubulin epitope tag,T7 gene 10 protein peptide tag, streptavidin tag, streptavidin bindingpeptide (SPB) tag, Strep-tag, Strep-tag II, albumin-binding protein(ABP) tag, alkaline phosphatase (AP) tag, bluetongue virus tag (B-tag),calmodulin binding peptide (CBP) tag, chloramphenicol acetyl transferase(CAT) tag, choline-binding domain (CBD) tag, chitin binding domain (CBD)tag, cellulose binding domain (CBP) tag, dihydrofolate reductase (DHFR)tag, galactose-binding protein (GBP) tag, maltose binding protein (MBP),glutathione-S-transferase (GST), Glu-Glu (EE) tag, human influenzahemagglutinin (HA) tag, horseradish peroxidase (HRP) tag, NE-tag, HSVtag, ketosteroid isomerase (KSI) tag, KT3 tag, LacZ tag, luciferase tag,NusA tag, PDZ domain tag, AviTag, Calmodulin-tag, E-tag, S-tag, SBP-tag,Softag 1, Softag 3, TC tag, VSV-tag, Xpress tag, Isopeptag, SpyTag,SnoopTag, Profinity eXact tag, Protein C tag, S1-tag, S-tag,biotin-carboxy carrier protein (BCCP) tag, green fluorescent protein(GFP) tag, small ubiquitin-like modifier (SUMO) tag, tandem affinitypurification (TAP) tag, HaloTag, Nus-tag, Thioredoxin-tag, Fc-tag, CYDtag, HPC tag, TrpE tag, ubiquitin tag, VSV-G epitope tag, V5 tag, and acombination thereof; optionally, wherein the first or second affinityacceptor peptide comprises two or more repeats of a tag sequence.

In some embodiments, the linker comprises a polynucleic acid sequenceencoding a cleavable linker. In some embodiments, the cleavable linkeris a ribosomal skipping site or an internal ribosomal entry site (IRES)element. In some embodiments, the ribosomal skipping site or IRES iscleaved when expressed in the cells. In some embodiments, the ribosomalskipping site is selected from the group consisting of F2A, T2A, P2A,and E2A. In some embodiments, the IRES element is selected from commoncellular or viral IRES sequences.

In some embodiments, the determining comprises performing biochemicalanalysis or mass spectrometry, such as tandem mass spectrometry. In someembodiments, the determining comprises obtaining a peptide sequence thatcorresponds to an MS/MS spectra of one or more peptides isolated fromthe enriched affinity acceptor tagged HLA-peptide complexes from apeptide database; wherein one or more sequences obtained identifies thesequence of the one or more peptides.

In some embodiments, the population of cells is a cell line selectedfrom HEK293T, expi293, HeLa, A375, 721.221, JEG-3, K562, Jurkat, Hep G2,SH-SY5Y, CACO-2, U937, U-2 OS, ExpiCHO, CHO and THP1. In someembodiments, the cell line is treated with one or more cytokines,checkpoint inhibitors, epigenetically-active drugs, IFN-γ, agents thatalter antigen processing (such as peptidase inhibitors, proteasomeinhibitors, and TAP inhibitors), or a combination thereof.

In some embodiments, the peptide database is a no-enzyme specificitypeptide database, such as a without modification database or a withmodification database. In some embodiments, the method further comprisessearching the peptide database using a reversed-database searchstrategy.

In some embodiments, the population of cells comprises at least 10⁵cells, at least 10⁶ cells or at least 10⁷ cells. In some embodiments,the population of cells is a population of dendritic cells, macrophages,cancer cells or B-cells. In some embodiments, the population of cellscomprises tumor cells. In some embodiments, the population of cells iscontacted with an agent prior to isolating said HLA-peptide complexesfrom the one or more cells. In some embodiments, said agent is aninflammatory cytokine, a chemical agent, an adjuvant, a therapeuticagent or radiation.

In some embodiments, the HLA allele is a mutated HLA allele.

In some embodiments, the sequence encoding the HLA allele comprises abarcode sequence. In some embodiments, the method further comprisesassaying for expression of the affinity acceptor tagged class I or classII HLA allele. In some embodiments, the assaying comprises assayingcomprises sequencing an affinity acceptor tagged class I or class II HLAallele, detecting affinity acceptor tagged class I or class II HLAallele RNA, detecting affinity acceptor tagged class I or class II HLAallele protein, or a combination thereof.

In some embodiments, the method comprises carrying out the steps of themethod for different HLA alleles. In some embodiments, each differentHLA allele comprises a unique barcode sequence. In some embodiments,each polynucleic acid encoding a different HLA allele comprises a uniquebarcode sequence.

Provided herein is a HLA-allele specific binding peptide sequencedatabase obtained by carrying out a method described herein. Providedherein is a combination of two or more HLA-allele specific bindingpeptide sequence databases obtained by carrying out a method describedherein repeatedly, each time using a different HLA-allele. Providedherein is a method for generating a prediction algorithm for identifyingHLA-allele specific binding peptides, comprising training a machine witha peptide sequence database described herein or a combination describedherein. In some embodiments, the machine combines one or more linearmodels, support vector machines, decision trees and neural networks. Insome embodiments, a variable used to train the machine comprises one ormore variables selected from the group consisting of peptide sequence,amino acid physical properties, peptide physical properties, expressionlevel of the source protein of a peptide within a cell, proteinstability, protein translation rate, ubiquitination sites, proteindegradation rate, translational efficiencies from ribosomal profiling,protein cleavability, protein localization, motifs of host protein thatfacilitate TAP transport, host protein is subject to autophagy, motifsthat favor ribosomal stalling, and protein features that favor NMD. Insome embodiments, the motifs that favor ribosomal stalling comprisespolyproline or polylysine stretches. In some embodiments, the proteinfeatures that favor NMD are selected from the group consisting of a long3′ UTR, a stop codon greater than 50nt upstream of last exon:exonjunction, and peptide cleavability. Provided herein is a method foridentifying HLA-allele specific binding peptides comprising analyzingthe sequence of a peptide with a machine which has been trained with apeptide sequence database obtained by carrying out a method describedherein for the HLA-allele. In some embodiments, the method comprisesdetermining the expression level of the source protein of the peptidewithin a cell; and wherein the source protein expression is a predictivevariable used by the machine. In some embodiments, the expression levelis determined by measuring the amount of source protein or the amount ofRNA encoding said source protein.

Provided herein is a composition comprising a recombinant polynucleicacid comprising two or more sequences each encoding an affinity acceptortagged HLA, wherein the sequences encoding the affinity acceptor taggedHLAs comprise (a) a sequence encoding a different recombinant HLA classI α-chain allele, (b) a sequence encoding an affinity acceptor peptide,and optionally, (c) a sequence encoding β2 microglobulin; wherein thesequences of (a) and (b), and optionally (c), are operatively linked.

Provided herein is a composition comprising a recombinant polynucleicacid comprising two or more sequences each comprising a sequenceencoding an affinity acceptor tagged HLA, wherein the sequences encodingthe affinity acceptor tagged HLAs comprise (a) a sequence encoding arecombinant HLA class II α-chain allele, (b) a sequence encoding anaffinity acceptor peptide, and optionally, (c) a sequence encoding a HLAclass II β-chain; wherein the sequences of (a) and (b), and optionally(c), are operatively linked. In some embodiments, the recombinantpolynucleic acid is isolated. In some embodiments, the class I HLA isselected from the group consisting of HLA-A, HLA-B, HLA-C, HLA-E, HLA-F,and HLA-G. In some embodiments, the class II HLA is selected from thegroup consisting of HLA-DR, HLA-DQ, and HLA-DP.

In some embodiments, the sequence encoding the affinity acceptor peptideis operatively linked to a sequence that encodes for an extracellularportion of the recombinant HLA allele. In some embodiments, the sequenceencoding the affinity acceptor molecule is operatively linked to theN-terminus of the sequence encoding the recombinant HLA allele. In someembodiments, the sequence encoding the affinity acceptor peptide isoperatively linked to a sequence encoding an intracellular portion ofthe recombinant HLA allele. In some embodiments, the sequence encodingthe affinity acceptor peptide is operatively linked to the C-terminus ofthe sequence encoding the recombinant HLA allele.

In some embodiments, the sequence encoding the affinity acceptor peptideis operatively linked to the sequence encoding the recombinant HLAallele by a linker.

In some embodiments, the two or more sequences encoding an affinityacceptor tagged HLA are expressed from the same polynucleotide. In someembodiments, the two or more sequences encoding an affinity acceptortagged HLA are expressed from different polynucleotides.

In some embodiments, the encoded affinity acceptor peptide bindsspecifically to an affinity acceptor peptide binding molecule.

In some embodiments, the two or more sequences encoding an affinityacceptor tagged HLA comprise two or more affinity acceptor peptides. Insome embodiments, the two or more sequences encoding an affinityacceptor tagged HLA comprise three or more sequences encoding anaffinity acceptor tagged HLA, wherein at least two of the three or moresequences encoding an affinity acceptor tagged HLA comprises the sameaffinity acceptor peptide. In some embodiments, the two or more affinityacceptor peptides are unique for each of the two or more sequencesencoding an affinity acceptor tagged HLA. In some embodiments, theencoded affinity acceptor peptide is selected from the group consistingof biotin acceptor peptide (BAP), poly-histidine tag,poly-histidine-glycine tag, poly-arginine tag, poly-aspartate tag,poly-cysteine tag, poly-phenylalanine, c-myc tag, Herpes simplex virusglycoprotein D (gD) tag, FLAG tag, KT3 epitope tag, tubulin epitope tag,T7 gene 10 protein peptide tag, streptavidin tag, streptavidin bindingpeptide (SPB) tag, Strep-tag, Strep-tag II, albumin-binding protein(ABP) tag, alkaline phosphatase (AP) tag, bluetongue virus tag (B-tag),calmodulin binding peptide (CBP) tag, chloramphenicol acetyl transferase(CAT) tag, choline-binding domain (CBD) tag, chitin binding domain (CBD)tag, cellulose binding domain (CBP) tag, dihydrofolate reductase (DHFR)tag, galactose-binding protein (GBP) tag, maltose binding protein (MBP),glutathione-S-transferase (GST), Glu-Glu (EE) tag, human influenzahemagglutinin (HA) tag, horseradish peroxidase (HRP) tag, NE-tag, HSVtag, ketosteroid isomerase (KSI) tag, KT3 tag, LacZ tag, luciferase tag,NusA tag, PDZ domain tag, AviTag, Calmodulin-tag, E-tag, S-tag, SBP-tag,Softag 1, Softag 3, TC tag, VSV-tag, Xpress tag, Isopeptag, SpyTag,SnoopTag, Profinity eXact tag, Protein C tag, S1-tag, S-tag,biotin-carboxy carrier protein (BCCP) tag, green fluorescent protein(GFP) tag, small ubiquitin-like modifier (SUMO) tag, tandem affinitypurification (TAP) tag, HaloTag, Nus-tag, Thioredoxin-tag, Fc-tag, CYDtag, HPC tag, TrpE tag, ubiquitin tag, VSV-G epitope tag, V5 tag, and acombination thereof; optionally, wherein the first or second affinityacceptor peptide comprises two or more repeats of a tag sequence. Insome embodiments, the affinity acceptor peptide binding molecule isbiotin or an antibody specific to the affinity acceptor peptide. In someembodiments, the affinity acceptor peptide binding molecule bindsspecifically to an affinity molecule. In some embodiments, the affinitymolecule is streptavidin, NeutrAvidin, or a derivative thereof. In someembodiments, the affinity acceptor peptide binding molecule does notspecifically interact with an amino acid sequence of the recombinantclass I or class II HLA.

In some embodiments, for two or more of the recombinant polynucleicacids: the sequence encoding the affinity acceptor tagged HLA is stablyintegrated into the genome of a cell.

In some embodiments, the sequence encoding β2 microglobulin or thesequence encoding the HLA class II β-chain is connected to a sequenceencoding a second affinity acceptor peptide. In some embodiments, thesecond affinity acceptor peptide comprises an HA tag. In someembodiments, the sequence encoding β2 microglobulin or the sequenceencoding the HLA class II β-chain is connected to the sequence encodingthe recombinant HLA and the affinity acceptor peptide by a linker. Insome embodiments, the linker comprises a polynucleic acid sequenceencoding a cleavable linker. In some embodiments, the cleavable linkeris a ribosomal skipping site or an internal ribosomal entry site (IRES)element. In some embodiments, the ribosomal skipping site or IRES iscleaved when expressed in the cells. In some embodiments, the ribosomalskipping site is selected from the group consisting of F2A, T2A, P2A,and E2A In some embodiments, the IRES element is selected from commoncellular or viral IRES sequences.

Provided herein is a composition comprising two or more isolatedpolypeptide molecules encoded by the polynucleic acid of a compositiondescribed herein. Provided herein is a composition comprising apopulation of cells comprising two or more polypeptide molecules encodedby the polynucleic acid of a composition described herein. Providedherein is a composition comprising a population of cells comprising acomposition described herein. Provided herein is a compositioncomprising a population of cells comprising one or more cells comprisinga composition described herein.

In some embodiments, the population of cells express one or moreendogenous class I or class II HLA alleles. In some embodiments, thepopulation of cells are engineered to lack one or more endogenous HLAclass I alleles. In some embodiments, the population of cells areengineered to lack endogenous HLA class I alleles. In some embodiments,the population of cells are engineered to lack one or more endogenousHLA class II alleles. In some embodiments, the population of cells areengineered to lack endogenous HLA class II alleles. In some embodiments,the population of cells are engineered to lack one or more endogenousHLA class I alleles and one or more endogenous HLA class II alleles.

In some embodiments, the population of cells is a population of low cellsurface HLA class I or class II expressing cells. In some embodiments,the composition is formulated using peptides or polynucleic acidsencoding peptides specific to an HLA type of a patient.

Provided herein is a method of making a cell comprising transducing ortransfecting two or more cells with the two or more polynucleic acids ofa composition described herein. Provided herein is a peptide identifiedaccording to a method described herein.

Provided herein is a method of inducing an anti-tumor response in amammal comprising administering to the mammal an effective amount of apolynucleic acid comprising a sequence of a peptide described herein.Provided herein is a method of inducing an anti-tumor response in amammal comprising administering to the mammal an effective amount of apeptide comprising the sequence of a peptide described herein. Providedherein is a method of inducing an anti-tumor response in a mammalcomprising administering to the mammal a cell comprising a peptidecomprising the sequence of a peptide described herein. Provided hereinis a method of inducing an anti-tumor response in a mammal comprisingadministering to the mammal a cell comprising an effective amount of apolynucleic acid comprising a sequence encoding a peptide comprising thesequence of a peptide described herein. In some embodiments, the cellpresents the peptide as an HLA-peptide complex. Provided herein is amethod of for inducing an immune response in a mammal comprisingadministering to the mammal an effective amount of a polynucleic acidcomprising a sequence encoding a peptide described herein. Providedherein is a method for inducing an immune response in a mammalcomprising administering to the mammal an effective amount of a peptidecomprising the sequence of a peptide described herein. Provided hereinis a method for inducing an immune response in a mammal comprisingadministering to the mammal an effective amount of a cell comprising apeptide comprising the sequence of a peptide described herein. Providedherein is a method for inducing an immune response in a mammalcomprising administering to the mammal an effective amount of a cellcomprising a polynucleic acid comprising a sequence encoding a peptidecomprising the sequence of a peptide described herein.

In some embodiments, the immune response is a T cell immune response. Insome embodiments, the immune response is a CD8 T cell response. In someembodiments, the immune response is a CD4 T cell response. In someembodiments, the immune response is humoral immune response.

Provided herein is a method for treating a mammal having a diseasecomprising administering to the mammal an effective amount of apolynucleic acid comprising a sequence encoding a peptide describedherein Provided herein is a method for treating a mammal having adisease comprising administering to the mammal an effective amount of apeptide comprising the sequence of a peptide described herein. Providedherein is a method for treating a mammal having a disease comprisingadministering to the mammal an effective amount of a cell comprising apeptide comprising the sequence of a peptide described herein. Providedherein is a method for treating a mammal having a disease comprisingadministering to the mammal an effective amount of a cell comprising apolynucleic acid comprising a sequence encoding a peptide comprising thesequence of a peptide described herein.

In some embodiments, the disease is cancer. In some embodiments, thedisease is infection by an infectious agent. In some embodiments, theinfectious agent is a pathogen, optionally a virus or bacteria, or aparasite. In some embodiments, the virus is selected from the groupconsisting of: BK virus (BKV), Dengue viruses (DENV-1, DENV-2, DENV-3,DENV-4, DENV-5), cytomegalovirus (CMV), Hepatitis B virus (HBV),Hepatitis C virus (HCV), Epstein-Barr virus (EBV), an adenovirus, humanimmunodeficiency virus (HIV), human T-cell lymphotrophic virus (HTLV-1),an influenza virus, RSV, HPV, rabies, mumps rubella virus, poliovirus,yellow fever, hepatitis A, hepatitis B, Rotavirus, varicella virus,human papillomavirus (HPV), smallpox, zoster, and any combinationthereof. In some embodiments, the bacteria is selected from the groupconsisting of: Klebsiella spp., Tropheryma whipplei, Mycobacteriumleprae, Mycobacterium lepromatosis, and Mycobacterium tuberculosis,typhoid, pneumococcal, meningococcal, haemophilus B, anthrax, tetanustoxoid, meningococcal group B, bcg, cholera, and any combinationthereof. In some embodiments, the parasite is a helminth or a protozoan.In some embodiments, the parasite is selected from the group consistingof: Leishmania spp., Plasmodium spp., Trypanosoma cruzi, Ascarislumbricoides, Trichuris trichiura, Necator americanus, Schistosoma spp.,and any combination thereof.

Provided herein is a method of enriching for immunogenic peptidescomprising: providing a population of cells comprising one or more cellsexpressing an affinity acceptor tagged HLA, wherein the affinityacceptor tagged HLA comprises an affinity acceptor peptide operativelylinked to a recombinant HLA encoded by a recombinant HLA allele; andenriching for HLA-peptide complexes comprising the affinity acceptortagged HLA. In some embodiments, the method further comprisesdetermining the sequence of immunogenic peptides isolated from theHLA-peptide complexes. In some embodiments, the determining comprisesusing LC-MS/MS.

Provided herein is a method of treating a disease or disorder in asubject, the method comprising administering to the subject an effectiveamount of a polynucleic acid comprising a sequence encoding a peptidedescribed herein. Provided herein is a method of treating a disease ordisorder in a subject, the method comprising administering to thesubject an effective amount of a peptide comprising the sequence of apeptide described herein. Provided herein is a method of treating adisease or disorder in a subject, the method comprising administering tothe subject an effective amount of a cell comprising a peptidecomprising the sequence of a peptide described herein. Provided hereinis a method of treating a disease or disorder in a subject, the methodcomprising administering to the subject a cell comprising an effectiveamount of a polynucleic acid comprising a sequence encoding a peptidecomprising the sequence of a peptide described herein.

Enrichment of HLA-Peptide Complexes

The genes encoding HLA class I and class II glycoproteins are amongstthe most polymorphic coding sequences in the human genome. However,there are relatively constant or invariable regions for each of the HLAclass I heavy chains and HLA class II α and β chains which can betargeted by antibodies to selectively capture any HLA class I heavychain or HLA class II α or β chain. However, since the α and β chainsare normally associated with each other in vivo, immunopurification ofthe α-chain of an intact soluble HLA can co-precipitate the β-chain andvice versa. Anti-HLA class II antibodies for the purpose of enrichingfor HLA-associated polypeptides can recognize conserved epitopespresented on either the α or β chain.

The enrichment method employing HLA allele specific antibodies orutilizing non-HLA specific reagents is well-known in the art. Forexample, HLA-C polypeptides are typically expressed by individuals atlower levels than HLA-A and HLA-B. Accordingly, in order to enhance thedetection of HLA-C using antibodies, it can be advantageous to provide aspecific immunopurification of HLA-C using an HLA-C specific antibody,in addition to other purification methods. Numerous examples ofmonoclonal or polyclonal antibodies which bind specifically toindividual HLA chains are commercially available.

Provided herein is a universal immunopurification (IP) pipeline forenriching one or more single allele HLA polypeptide complexes.Illustrative of such a method for enriching for an HLA-associatedpolypeptide is a method which comprises an immunopurification step.Universal IP pipeline comprises universal IP constructs consisting of aDNA construct coding for affinity-tagged HLA class I or class II allelesthat are expressed off an expression vector via cellular transfection ortransduction. Non-limiting example of an expression vector is alentiviral vector.

Cells transfected or transduced with universal IP constructs were eitherexpanded or selected and then expanded prior to LC-MS/MS sequenceanalyses. Suitable cell populations for transfection or transductioninclude, e.g., class I deficient cells lines in which a single HLA classI allele is expressed, class II deficient cell lines in which a singlepair of HLA class II alleles are expressed, or class I and class IIdeficient cell lines in which a single HLA class I and/or single pair ofclass II alleles are expressed. As an exemplary embodiment, the class Ideficient B cell line is B721.221. In some embodiments, the cells areA375, or HEK293T, HeLa, or expi293. However, it is clear to a skilledperson that other cell populations can be generated which are class Iand/or class II deficient. Methods for generating class I and/or classII deficient cells as well as class I and/or class II deficient celllines are known in the art, and an exemplary method fordeleting/inactivating endogenous class I or class II genes includesCRISPR-Cas9 mediated genome editing in, for example, THP-1 cells. Insome embodiments, the populations of cells are professional antigenpresenting cells, such as macrophages, B cells, and dendritic cells. Thecells can be B cells or dendritic cells. In some embodiments, the cellsare tumor cells or cells from a tumor cell line. In some embodiments,the cells are cells isolated from a patient. In some embodiments, thecells contain an infectious agent or a portion thereof.

In some embodiments, universal IP constructs comprise class I or classII HLA constructs comprising an affinity acceptor tag and affinitymolecule. In some embodiments, universal IP constructs comprise at leastone specifically binding affinity acceptor tag and affinity molecule. Insome embodiments, an affinity acceptor tag is poly-histidine tag,poly-histidine-glycine tag, poly-arginine tag, poly-aspartate tag,poly-cysteine tag, poly-phenylalanine, c-myc tag, Herpes simplex virusglycoprotein D (gD) tag, FLAG tag, KT3 epitope tag, tubulin epitope tag,T7 gene 10 protein peptide tag, streptavidin tag, streptavidin bindingpeptide (SPB) tag, Strep-tag, Strep-tag II, albumin-binding protein(ABP) tag, alkaline phosphatase (AP) tag, bluetongue virus tag (B-tag),calmodulin binding peptide (CBP) tag, chloramphenicol acetyl transferase(CAT) tag, choline-binding domain (CBD) tag, chitin binding domain (CBD)tag, cellulose binding domain (CBP) tag, dihydrofolate reductase (DHFR)tag, galactose-binding protein (GBP) tag, maltose binding protein (MBP),glutathione-S-transferase (GST), Glu-Glu (EE) tag, human influenzahemagglutinin (HA) tag, horseradish peroxidase (HRP) tag, NE-tag, HSVtag, ketosteroid isomerase (KSI) tag, KT3 tag, LacZ tag, luciferase tag,NusA tag, PDZ domain tag, AviTag, Calmodulin-tag, E-tag, S-tag, SBP-tag,Softag 1, Softag 3, TC tag, VSV-tag, Xpress tag, Isopeptag, SpyTag,SnoopTag, Profinity eXact tag, Protein C tag, S1-tag, S-tag,biotin-carboxy carrier protein (BCCP) tag, green fluorescent protein(GFP) tag, small ubiquitin-like modifier (SUMO) tag, tandem affinitypurification (TAP) tag, HaloTag, Nus-tag, Thioredoxin-tag, Fc-tag, CYDtag, HPC tag, TrpE tag, ubiquitin tag, a VSV-G epitope tag derived fromthe Vesicular Stomatis viral glycoprotein, or a V5 tag derived from asmall epitope (Pk) found on the P and V proteins of the paramyxovirus ofsimian virus 5 (SV5). In some embodiments, the affinity acceptor tag caninclude multiple repeats of the tag sequence (e.g. 3× poly histidinetag, 3× FLAG tag). In some embodiments, the affinity acceptor tag caninclude multiple repeats of the tag sequence (e.g. 3× poly histidinetag, 3× FLAG tag). In some embodiments, the affinity acceptor tag is an“epitope tag,” which is a type of peptide tag that adds a recognizableepitope (antibody binding site) to the HLA-protein to provide binding ofcorresponding antibody, thereby allowing identification or affinitypurification of the tagged protein. Non-limiting example of an epitopetag is protein A or protein G, which binds to IgG. In some embodiments,affinity acceptor tags include the biotin acceptor peptide (BAP) orHuman influenza hemagglutinin (HA) peptide sequence. Numerous other tagmoieties are known to, and can be envisioned by, the ordinarily skilledartisan, and are contemplated herein. Any peptide tag can be used aslong as it is capable of being expressed as an element of an affinityacceptor tagged HLA-peptide complex.

The affinity tags can be placed on either the N-terminus or C-terminusof the HLA allele. A cleavage sequence, such as F2A, or an internalribosome entry site (IRES) can be placed between the α-chain andβ2-microglobulin (class I) or between the α-chain and β-chain (classII). In some embodiments, a single class I HLA allele is HLA-A*02:01,HLA-A*23:01 and HLA-B*14:02, or HLA-E*01:01, and class II HLA allele isHLA-DRB*01:01, HLA-DRB*01:02 and HLA-DRB*11:01, HLA-DRB*15:01, orHLA-DRB*07:01. In some embodiments, the cleavage sequence is a T2A, P2A,E2A, or F2A sequence. For example, the cleavage sequence can be E G R GS L L T C G D V E E N P G P (T2A), A T N F S L L K Q A G D V E E N P G P(P2A), Q C T N Y A L L K L A G D V E S N P G P (E2A), or V K Q T L N F DL L K L A G D V E S N P G P (F2A).

In some embodiments, HLA-peptide complex immunopurification isbiotin-based. In some embodiments, HLA-peptide compleximmunopurification is streptavidin or NeutrAvidin based. In someembodiments, HLA-peptide complexes can also be enriched from abiological sample by chromatography techniques, such as HPLC. In someembodiments, the depletion of high abundance serum proteins can be usedto enrich for HLA-peptide complexes. In some embodiments, methods forremoving abundant serum proteins include dye ligands (for albumin),protein A and G (for γ-globulins) or specific antibodies which bind withhigh affinity and selectively deplete these species from the sample(Govorukhina, Reijmers et al. 2006). Such strategies would increase thenumber of HLA-derived peptide sequences identified in a single massspectrometry analysis.

The degree of enrichment desirable to optimize the resolution ofparticular HLA sequences from a biological sample will depend on theinitial concentration of the HLA sequence in the biological sample, andthe concentration and nature of other non-HLA proteins in the sample.

To enrich HLA-peptide complexes within a biological sample, classicalprotein purification techniques can be used alone or in combination withthe universal IP pipeline methods provided herein. Classical proteinseparation (purification) techniques are based on; size differences(ultrafiltration, gel filtration, or size exclusion chromatography);charge differences (pi) (anion/cation exchange chromatography, orhydrophobic interaction chromatography); and combinations of size andcharge differences (1D or 2D electrophoresis). Immunopurificationoptions include the use of monoclonal or polyclonal antibodies thatspecifically bind HLA proteins. Other protein affinity purificationoptions involve the use of proteins that are known to bind HLA, theseinclude; CD8, which binds to the α3 domain of all HLA class I proteins;CD4 which binds to all HLA class II proteins; autologous T-cellreceptors; and antigenic peptides which bind HLA with high affinity(computer modelling algorithms can be used to predict peptide/HLAbinding characteristics). Any of these high HLA affinity protein optionscan be immobilized onto an insoluble solid support to prepare anaffinity matrix which can be used to capture the HLA from a liquidbiological sample. Appropriate elution conditions will result in theconcentration and purification (isolation) of the sample's HLA content.

In some embodiments, the enriching comprises enriching for intact cellsexpressing the affinity acceptor tagged HLA-peptide complexes. In someembodiments, the method does not comprise lysing the cells before theenriching. In some embodiments, the method further comprises lysing theone or more cells before the enriching. In some embodiments, theenriching comprises contacting an affinity acceptor peptide bindingmolecule to the affinity acceptor tagged HLA-peptide complexes, whereinthe affinity acceptor peptide binding molecule binds specifically to theaffinity acceptor peptide. In some instances, the enriching does notcomprise use of a tetramer reagent.

Disease Specific Antigens

In some embodiments, the size of at least one antigenic peptide moleculecan comprise, but is not limited to, about 8, about 9, about 10, about11, about 12, about 13, about 14, about 15, about 16, about 17, about18, about 19, about 20, about 21, about 22, about 23, about 24, about25, about 26, about 27, about 28, about 29, about 30, about 31, about32, about 33, about 34, about 35, about 36, about 37, about 38, about39, about 40, about 41, about 42, about 43, about 44, about 45, about46, about 47, about 48, about 49, about 50, about 60, about 70, about80, about 90, about 100, about 110, about 120 or greater amino moleculeresidues, and any range derivable therein.

In some embodiments, the antigenic peptide molecules are equal to orless than 50 amino acids. In some embodiments, the antigenic peptidemolecules are equal to about 20 to about 30 amino acids. A longerpeptide can be designed in several ways. For example, when theHLA-binding regions are predicted or known, a longer peptide can consistof either: individual binding peptides with an extension of 0-10 aminoacids toward the N- and C-terminus of each corresponding gene product. Alonger peptide can also consist of a concatenation of some or all of thebinding peptides with extended sequences for each. In another case, whensequencing reveals a long (>10 residues) epitope sequence present in thediseased tissue (e.g. due to a frameshift, read-through or introninclusion that leads to a novel peptide sequence), a longer peptide canconsist of the entire stretch of novel disease-specific amino acids. Inboth cases, use of a longer peptide requires endogenous processing byprofessional antigen presenting cells such as dendritic cells and canlead to more effective antigen presentation and induction of T cellresponses. In some embodiments, the extended sequence is altered toimprove the biochemical properties of the polypeptide (properties suchas solubility or stability) or to improve the likelihood for efficientproteasomal processing of the peptide.

The antigenic peptides and polypeptides can bind an HLA protein. In someembodiments, the antigenic peptides can bind an HLA protein with greateraffinity than a corresponding native/wild-type peptide. The antigenicpeptide can have an IC50 of about less than 1000 nM, about less than 500nM, about less than 250 nM, about less than 200 nM, about less than 150nM, about less than 100 nM, or about less than 50 nM. In someembodiments, the antigenic peptides do not induce an autoimmune responseand/or invoke immunological tolerance when administered to a subject.

The present disclosure also provides compositions comprising a pluralityof antigenic peptides. Reference to antigenic peptides includes anysuitable delivery modality that can result in introduction of thepeptide into a subject's cell (e.g., nucleic acid). In some embodiments,the composition comprises at least 3 or more antigenic peptides. In someembodiments the composition contains at least about 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, 35, 40, 45, or 50 distinct peptides. In some embodiments thecomposition contains at least 20 distinct peptides. In some embodimentsthe composition contains at most 20 distinct peptides. According to thepresent disclosure, 2 or more of the distinct peptides can be derivedfrom the same polypeptide. For example, if an antigenic mutation encodesa polypeptide, two or more of the antigenic peptides can be derived fromthe polypeptide. In one embodiment, the two or more antigenic peptidesderived from the polypeptide can comprise a tiled array that spans thepolypeptide (e.g., the antigenic peptides can comprise a series ofoverlapping antigenic peptides that spans a portion, or all, of thepolypeptide). Antigenic peptides can be derived from any protein codinggene. The antigenic peptides can be derived from mutations in humancancer or from an infectious agent or an autoimmune disease.

The antigenic peptides, polypeptides, and analogs can be furthermodified to contain additional chemical moieties not normally part ofthe protein. Those derivatized moieties can improve the solubility, thebiological half-life, absorption of the protein, or binding affinity.The moieties can also reduce or eliminate any desirable side effects ofthe proteins and the like. An overview for those moieties can be foundin Remington's Pharmaceutical Sciences, 20th ed., Mack Publishing Co.,Easton, Pa. (2000). For example, antigenic peptides and polypeptideshaving the desired activity can be modified as necessary to providecertain desired attributes, e.g. improved pharmacologicalcharacteristics, while increasing or at least retaining substantiallyall of the biological activity of the unmodified peptide to bind thedesired MHC molecule and activate the appropriate T cell. For instance,the antigenic peptide and polypeptides can be subject to variouschanges, such as substitutions, either conservative or non-conservative,where such changes might provide for certain advantages in their use,such as improved MHC binding. Such conservative substitutions canencompass replacing an amino acid residue with another amino acidresidue that is biologically and/or chemically similar, e.g., onehydrophobic residue for another, or one polar residue for another. Theeffect of single amino acid substitutions can also be probed usingD-amino acids. Such modifications can be made using well known peptidesynthesis procedures, as described in e.g., Merrifield, Science232:341-347 (1986), Barany & Merrifield, The Peptides, Gross &Meienhofer, eds. (N.Y., Academic Press), pp. 1-284 (1979); and Stewart &Young, Solid Phase Peptide Synthesis, (Rockford, III., Pierce), 2d Ed.(1984).

The antigenic peptide can also be modified by extending or decreasingthe compound's amino acid sequence, e.g., by the addition or deletion ofamino acids. The antigenic peptides, polypeptides, or analogs can alsobe modified by altering the order or composition of certain residues. Itwill be appreciated by the skilled artisan that certain amino acidresidues essential for biological activity, e.g., those at criticalcontact sites or conserved residues, may generally not be alteredwithout an adverse effect on biological activity. The non-critical aminoacids need not be limited to those naturally occurring in proteins, suchas L-a-amino acids, or their D-isomers, but can include non-naturalamino acids as well, such as β-γ-δ-amino acids, as well as manyderivatives of L-a-amino acids.

An antigen peptide can be optimized by using a series of peptides withsingle amino acid substitutions to determine the effect of electrostaticcharge, hydrophobicity, etc. on MHC binding. For instance, a series ofpositively charged (e.g., Lys or Arg) or negatively charged (e.g., Glu)amino acid substitutions can be made along the length of the peptiderevealing different patterns of sensitivity towards various MHCmolecules and T cell receptors. In addition, multiple substitutionsusing small, relatively neutral moieties such as Ala, Gly, Pro, orsimilar residues can be employed. The substitutions can behomo-oligomers or hetero-oligomers. The number and types of residueswhich are substituted or added depend on the spacing necessary betweenessential contact points and certain functional attributes which aresought (e.g., hydrophobicity versus hydrophilicity). Increased bindingaffinity for an MHC molecule or T cell receptor can also be achieved bysuch substitutions, compared to the affinity of the parent peptide. Inany event, such substitutions should employ amino acid residues or othermolecular fragments chosen to avoid, for example, steric and chargeinterference which might disrupt binding. Amino acid substitutions aretypically of single residues. Substitutions, deletions, insertions orany combination thereof can be combined to arrive at a final peptide.

An antigenic peptide can be modified to provide desired attributes. Forinstance, the ability of the peptides to induce CTL activity can beenhanced by linkage to a sequence which contains at least one epitopethat is capable of inducing a T helper cell response. In someembodiments, immunogenic peptides/T helper conjugates are linked by aspacer molecule. In some embodiments, a spacer comprises relativelysmall, neutral molecules, such as amino acids or amino acid mimetics,which are substantially uncharged under physiological conditions.Spacers can be selected from, e.g., Ala, Gly, or other neutral spacersof nonpolar amino acids or neutral polar amino acids. It will beunderstood that the optionally present spacer need not be comprised ofthe same residues and thus can be a hetero- or homo-oligomer. Theantigenic peptide can be linked to the T helper peptide either directlyor via a spacer either at the amino or carboxy terminus of the peptide.The amino terminus of either the antigenic peptide or the T helperpeptide can be acylated. Exemplary T helper peptides include tetanustoxoid 830-843, influenza 307-319, malaria circumsporozoite 382-398 and378-389.

Mono-Allelic HLA Cell Lines

A mono-allelic cell line expressing either a single class I HLA allele,a single pair of class II HLA alleles, or a single class I HLA alleleand a single pair of class II HLA alleles can be generated bytransducing or transfecting a suitable cell population with apolynucleic acid, e.g., a vector, coding a single HLA allele. Suitablecell populations include, e.g., class I deficient cells lines in which asingle HLA class I allele is expressed, class II deficient cell lines inwhich a single pair of HLA class II alleles are expressed, or class Iand class II deficient cell lines in which a single HLA class I and/orsingle pair of class II alleles are expressed. As an exemplaryembodiment, the class I deficient B cell line is B721.221. However, itis clear to a skilled person that other cell populations can begenerated which are class I and/or class II deficient. An exemplarymethod for deleting/inactivating endogenous class I or class II genesincludes CRISPR-Cas9 mediated genome editing in, for example, THP-1cells. In some embodiments, the populations of cells are professionalantigen presenting cells, such as macrophages, B cells, and dendriticcells. The cells can be B cells or dendritic cells. In some embodiments,the cells are tumor cells or cells from a tumor cell line. In someembodiments, the cells are cells isolated from a patient. In someembodiments, the cells contain an infectious agent or a portion thereof.In some embodiments, the population of cells comprises at least 10⁷cells. In some embodiments, the population of cells are furthermodified, such as by increasing or decreasing the expression and/oractivity of at least one gene. In some embodiments, the gene encodes amember of the immunoproteasome. The immunoproteasome is known to beinvolved in the processing of HLA class I binding peptides and includesthe LMP2 (β1i), MECL-1 (β2i), and LMP7 (β5i) subunits. Theimmunoproteasome can also be induced by interferon-gamma. Accordingly,in some embodiments, the population of cells can be contacted with oneor more cytokines, growth factors, or other proteins. The cells can bestimulated with inflammatory cytokines such as interferon-gamma, IL-10,IL-6, and/or TNF-α. The population of cells can also be subjected tovarious environmental conditions, such as stress (heat stress, oxygendeprivation, glucose starvation, DNA damaging agents, etc.). In someembodiments, the cells are contacted with one or more of a chemotherapydrug, radiation, targeted therapies, immunotherapy. The methodsdisclosed herein can therefore be used to study the effect of variousgenes or conditions on HLA peptide processing and presentation. In someembodiments, the conditions used are selected so as to match thecondition of the patient for which the population of HLA-peptides is tobe identified.

A single HLA-allele of the present disclosure can be encoded andexpressed using a viral based system (e.g., an adenovirus system, anadeno associated virus (AAV) vector, a poxvirus, or a lentivirus).Plasmids that can be used for adeno associated virus, adenovirus, andlentivirus delivery have been described previously (see e.g., U.S. Pat.Nos. 6,955,808 and 6,943,019, and U.S. Patent application No.20080254008, hereby incorporated by reference). Among vectors that canbe used in the practice of the present disclosure, integration in thehost genome of a cell is possible with retrovirus gene transfer methods,often resulting in long term expression of the inserted transgene. In anexemplary embodiment, the retrovirus is a lentivirus. Additionally, hightransduction efficiencies have been observed in many different celltypes and target tissues. The tropism of a retrovirus can be altered byincorporating foreign envelope proteins, expanding the potential targetpopulation of target cells. A retrovirus can also be engineered to allowfor conditional expression of the inserted transgene, such that onlycertain cell types are infected by the lentivirus. Cell type specificpromoters can be used to target expression in specific cell types.Lentiviral vectors are retroviral vectors (and hence both lentiviral andretroviral vectors can be used in the practice of the presentdisclosure). Moreover, lentiviral vectors are able to transduce orinfect non-dividing cells and typically produce high viral titers. Anexemplary lentiviral vector that can be used to generate stable celllines transduced to express HLA class I and class II is shown in FIG. 3.

Selection of a retroviral gene transfer system can depend on the targettissue. Retroviral vectors are comprised of cis-acting long terminalrepeats with packaging capacity for up to 6-10 kb of foreign sequence.The minimum cis-acting LTRs are sufficient for replication and packagingof the vectors, which are then used to integrate the desired nucleicacid into the target cell to provide permanent expression. Widely usedretroviral vectors that can be used in the practice of the presentdisclosure include those based upon murine leukemia virus (MuLV), gibbonape leukemia virus (GaLV), Simian Immunodeficiency virus (SIV), humanimmunodeficiency virus (HIV), and combinations thereof (see, e.g.,Buchscher et al., (1992) J. Virol. 66:2731-2739; Johann et al., (1992)J. Virol. 66:1635-1640; Sommnerfelt et al., (1990) Virol. 176:58-59;Wilson et al., (1998) J. Virol. 63:2374-2378; Miller et al., (1991) J.Virol. 65:2220-2224; PCT/US94/05700). Also, useful in the practice ofthe present disclosure is a minimal non-primate lentiviral vector, suchas a lentiviral vector based on the equine infectious anemia virus(EIAV) (see, e.g., Balagaan, (2006) J Gene Med; 8: 275-285, Publishedonline 21 Nov. 2005 in Wiley InterScience DOI: 10.1002/jgm.845). Thevectors can have cytomegalovirus (CMV) promoter driving expression ofthe target gene. Accordingly, the present disclosure contemplatesamongst vector(s) useful in the practice of the present disclosure:viral vectors, including retroviral vectors and lentiviral vectors.

Any HLA allele can be expressed in the cell population. In an exemplaryembodiment, the HLA allele is a class I HLA allele. In some embodiments,the class I HLA allele is an HLA-A allele or an HLA-B allele. In someembodiments, the HLA allele is a class II HLA allele. Sequences of classI and class II HLA alleles can be found in the IPD-IMGT/HLA Database.Exemplary HLA alleles include, but are not limited to, HLA-A*02:01,HLA-B*14:02, HLA-A*23:01, HLA-E*01:01, HLA-DRB*01:01, HLA-DRB*01:02,HLA-DRB*11:01, HLA-DRB*15:01, and HLA-DRB*07:01.

In some embodiments, the HLA allele is selected so as to correspond to agenotype of interest. In some embodiments, the HLA allele is a mutatedHLA allele, which can be non-naturally occurring allele or a naturallyoccurring allele in an afflicted patient. The methods disclosed hereinhave the further advantage of identifying HLA binding peptides for HLAalleles associated with various disorders as well as alleles which arepresent at low frequency. Accordingly, in some embodiments, method theHLA allele is present at a frequency of less than 1% within apopulation, such as within the Caucasian population.

In some embodiments, the nucleic acid sequence encoding the HLA allelefurther comprises an affinity acceptor tag which can be used toimmunopurify the HLA-protein. Suitable tags are well-known in the art.In some embodiments, an affinity acceptor tag is poly-histidine tag,poly-histidine-glycine tag, poly-arginine tag, poly-aspartate tag,poly-cysteine tag, poly-phenylalanine, c-myc tag, Herpes simplex virusglycoprotein D (gD) tag, FLAG tag, KT3 epitope tag, tubulin epitope tag,T7 gene 10 protein peptide tag, streptavidin tag, streptavidin bindingpeptide (SPB) tag, Strep-tag, Strep-tag II, albumin-binding protein(ABP) tag, alkaline phosphatase (AP) tag, bluetongue virus tag (B-tag),calmodulin binding peptide (CBP) tag, chloramphenicol acetyl transferase(CAT) tag, choline-binding domain (CBD) tag, chitin binding domain (CBD)tag, cellulose binding domain (CBP) tag, dihydrofolate reductase (DHFR)tag, galactose-binding protein (GBP) tag, maltose binding protein (MBP),glutathione-S-transferase (GST), Glu-Glu (EE) tag, human influenzahemagglutinin (HA) tag, horseradish peroxidase (HRP) tag, NE-tag, HSVtag, ketosteroid isomerase (KSI) tag, KT3 tag, LacZ tag, luciferase tag,NusA tag, PDZ domain tag, AviTag, Calmodulin-tag, E-tag, S-tag, SBP-tag,Softag 1, Softag 3, TC tag, VSV-tag, Xpress tag, Isopeptag, SpyTag,SnoopTag, Profinity eXact tag, Protein C tag, S1-tag, S-tag,biotin-carboxy carrier protein (BCCP) tag, green fluorescent protein(GFP) tag, small ubiquitin-like modifier (SUMO) tag, tandem affinitypurification (TAP) tag, HaloTag, Nus-tag, Thioredoxin-tag, Fc-tag, CYDtag, HPC tag, TrpE tag, ubiquitin tag, a VSV-G epitope tag derived fromthe Vesicular Stomatis viral glycoprotein, or a V5 tag derived from asmall epitope (Pk) found on the P and V proteins of the paramyxovirus ofsimian virus 5 (SV5). In some embodiments, the affinity acceptor tag isan “epitope tag,” which is a type of peptide tag that adds arecognizable epitope (antibody binding site) to the HLA-protein toprovide binding of corresponding antibody, thereby allowingidentification or affinity purification of the tagged protein.Non-limiting example of an epitope tag is protein A or protein G, whichbinds to IgG. In some embodiments, affinity acceptor tags include thebiotin acceptor peptide (BAP) or Human influenza hemagglutinin (HA)peptide sequence. Numerous other tag moieties are known to, and can beenvisioned by, the ordinarily skilled artisan, and are contemplatedherein. Any peptide tag can be used as long as it is capable of beingexpressed as an element of an affinity acceptor tagged HLA-peptidecomplex.

The methods provided herein comprise isolating HLA-peptide complexesfrom the cells transfected or transduced with universal IP HLAconstructs. In some embodiments, the complexes can be isolated usingstandard immunoprecipitation techniques known in the art withcommercially available antibodies. The cells can be first lysed. HLAclass I-peptide complexes can be isolated using HLA class I specificantibodies such as the W6/32 antibody, while HLA class II-peptidecomplexes can be isolated using HLA class II specific antibodies such asthe M5/114.15.2 monoclonal antibody. In some embodiments, the single (orpair of) HLA alleles are expressed as a fusion protein with a peptidetag and the HLA-peptide complexes are isolated using binding moleculesthat recognize the peptide tags.

The methods further comprise isolating peptides from said HLA-peptidecomplexes and sequencing the peptides. The peptides are isolated fromthe complex by any method known to one of skill in the art, such as acidelution. While any sequencing method can be used, methods employing massspectrometry, such as liquid chromatography-mass spectrometry (LC-MS orLC-MS/MS, or alternatively HPLC-MS or HPLC-MS/MS) are utilized in someembodiments. These sequencing methods are well-known to a skilled personand are reviewed in Medzihradszky K F and Chalkley R J. Mass SpectromRev. 2015 January-February; 34(1):43-63.

In some embodiments, the population of cells expresses one or moreendogenous HLA alleles. In some embodiments, the population of cells isan engineered population of cells lacking one or more endogenous HLAclass I alleles. In some embodiments, the population of cells is anengineered population of cells lacking endogenous HLA class I alleles.In some embodiments, the population of cells is an engineered populationof cells lacking one or more endogenous HLA class II alleles. In someembodiments, the population of cells is an engineered population ofcells lacking endogenous HLA class II alleles or an engineeredpopulation of cells lacking endogenous HLA class I alleles andendogenous HLA class II alleles. In some embodiments, the population ofcells comprises cells that have been enriched or sorted, such as byfluorescence activated cell sorting (FACS). In some embodiments,fluorescence activated cell sorting (FACS) is used to sort thepopulation of cells. In some embodiments, the population of cells ispreviously FACS sorted for cell surface expression of either class I orclass II HLA or both class I and class II HLA. For example, FACS can beused to sort the population of cells for cell surface expression of anHLA class I allele, an HLA class II allele, or a combination thereof.

Libraries of Affinity Acceptor Tagged HLA Constructs

The term “library” as used herein refers to a collection of nucleic acidmolecules (circular or linear). In one embodiment, a library cancomprise a plurality (i.e., two or more) of nucleic acid molecules,which can be from a common source organism, organ, tissue, or cell. Insome embodiments, a library is representative of all or a portion or asignificant portion of the nucleic acid content of an organism (a“genomic” library), or a set of nucleic acid molecules representative ofall or a portion or a significant portion of the expressed nucleic acidmolecules (a cDNA library or segments derived therefrom) in a cell,tissue, organ or organism. A library can also comprise random sequencesmade by de novo synthesis, mutagenesis of one or more sequences and thelike. Such libraries can be contained in one or more vectors. A libraryof affinity acceptor tagged HLA constructs as provided herein comprisesa DNA sequence encoding elements of a HLA allele, an affinity acceptorpeptide, or a linker. Appropriate molecular biological techniques can befound in Sambrook et al. (Molecular Cloning; A Laboratory Manual, NewYork: Cold Spring Harbor Laboratory Press, 1989). Several methods forfacilitating the cloning of nucleic acid segments have been described,e.g., as in the following references: Ferguson, J., et al., Gene 16:191(1981) and Hashimoto-Gotoh, T., et al., Gene 41:125 (1986). Other termsused in the fields of recombinant nucleic acid technology and molecularand cell biology as used herein will be generally understood by one ofordinary skill in the applicable arts.

The various elements or domains of a recombinant HLA allele can bearranged in any order between the N-terminal and C-terminal ends of therecombinant HLA allele. An element or domain that is closer to theN-terminus of a recombinant polypeptide encoded from a recombinant HLAallele than another element or domain is said to be “N-terminal” of theother element or domain. Similarly, an element or domain that is closerto the C-terminus of a recombinant polypeptide encoded from arecombinant HLA allele than another element or domain is said to be“C-terminal” of the other element or domain. Unless expressly statedotherwise, different elements or domains of a recombinant polypeptideencoded from a recombinant HLA allele need not be adjacent (that is,without one or more intervening elements or domains). In someembodiments, different elements or domains of a recombinant polypeptideencoded from a recombinant HLA allele can be adjacent.

A recombinant polypeptide encoded from a recombinant HLA allele caninclude one or more optional elements, such as one or more linker(s),peptide tags (such as, epitope tags), or protease-recognition site(s).In some embodiments, a peptide tag is an affinity acceptor peptide. Alinker is a relatively short series of amino acids that separates otherelements or domains of the recombinant protein. In some embodiments, alinker is from 1 to 100 amino acids in length; for example, from 5 to75, from 10 to 60, from 15 to 50, from 15 to 40, or from 1 to 50 aminoacids in length.

Methods of expressing proteins in heterologous expression systems arewell known in the art. Typically, a nucleic acid molecule encoding allor part of a protein of interest (such as a recombinant HLA class I orclass II affinity acceptor tagged peptide) is obtained using methodssuch as those described herein. The protein-encoding nucleic acidsequence is cloned into an expression vector that is suitable for theparticular host cell of interest using standard recombinant DNAprocedures. Expression vectors include (among other elements) regulatorysequences (e.g., promoters) that can be operably linked to the desiredprotein-encoding nucleic acid molecule to cause the expression of suchnucleic acid molecule in the host cell. Together, the regulatorysequences and the protein-encoding nucleic acid sequence are an“expression cassette.” Expression vectors can also include an origin ofreplication, marker genes that provide phenotypic selection intransformed cells, one or more other promoters, and a polylinker regioncontaining several restriction sites for insertion of heterologousnucleic acid sequences.

Expression vectors useful for expression of heterologous protein(s) in amultitude of host cells are well known in the art, and some specificexamples are provided herein. The host cell is transfected with (orinfected with a virus containing) the expression vector using any methodsuitable for the particular host cell. Such transfection methods arealso well known in the art and non-limiting exemplar methods aredescribed herein. The transfected or transduced host cell is capable ofexpressing the protein encoded by the corresponding nucleic acidsequence in the expression cassette.

In some embodiments, class I or class II HLA constructs comprising anaffinity acceptor tag and affinity molecule at N-terminus or C-terminus.In some embodiments, class I or class II HLA constructs comprise atleast one specifically binding affinity acceptor tag and affinitymolecule. In some embodiments, an affinity acceptor tag ispoly-histidine tag, poly-histidine-glycine tag, poly-arginine tag,poly-aspartate tag, poly-cysteine tag, poly-phenylalanine, c-myc tag,Herpes simplex virus glycoprotein D (gD) tag, FLAG tag, KT3 epitope tag,tubulin epitope tag, T7 gene 10 protein peptide tag, streptavidin tag,streptavidin binding peptide (SPB) tag, Strep-tag, Strep-tag II,albumin-binding protein (ABP) tag, alkaline phosphatase (AP) tag,bluetongue virus tag (B-tag), calmodulin binding peptide (CBP) tag,chloramphenicol acetyl transferase (CAT) tag, choline-binding domain(CBD) tag, chitin binding domain (CBD) tag, cellulose binding domain(CBP) tag, dihydrofolate reductase (DHFR) tag, galactose-binding protein(GBP) tag, maltose binding protein (MBP), glutathione-S-transferase(GST), Glu-Glu (EE) tag, human influenza hemagglutinin (HA) tag,horseradish peroxidase (HRP) tag, NE-tag, HSV tag, ketosteroid isomerase(KSI) tag, KT3 tag, LacZ tag, luciferase tag, NusA tag, PDZ domain tag,AviTag, Calmodulin-tag, E-tag, S-tag, SBP-tag, Softag 1, Softag 3, TCtag, VSV-tag, Xpress tag, Isopeptag, SpyTag, SnoopTag, Profinity eXacttag, Protein C tag, S1-tag, S-tag, biotin-carboxy carrier protein (BCCP)tag, green fluorescent protein (GFP) tag, small ubiquitin-like modifier(SUMO) tag, tandem affinity purification (TAP) tag, HaloTag, Nus-tag,Thioredoxin-tag, Fc-tag, CYD tag, HPC tag, TrpE tag, ubiquitin tag, aVSV-G epitope tag derived from the Vesicular Stomatis viralglycoprotein, or a V5 tag derived from a small epitope (Pk) found on theP and V proteins of the paramyxovirus of simian virus 5 (SV5). In someembodiments, the affinity acceptor tag can include multiple repeats ofthe tag sequence (e.g. 3× poly histidine tag, 3× FLAG tag). In someembodiments, the affinity acceptor tag can include multiple repeats ofthe tag sequence (e.g. 3× poly histidine tag, 3× FLAG tag). In someembodiments, the affinity acceptor tag is an “epitope tag,” which is atype of peptide tag that adds a recognizable epitope (antibody bindingsite) to the HLA-protein to provide binding of corresponding antibody,thereby allowing identification or affinity purification of the taggedprotein. Non-limiting example of an epitope tag is protein A or proteinG, which binds to IgG.

In some embodiments, affinity acceptor tags include the biotin acceptorpeptide (BAP) or Human influenza hemagglutinin (HA) peptide sequence.Numerous other tag moieties are known to, and can be envisioned by, theordinarily skilled artisan, and are contemplated herein. Any peptide tagcan be used as long as it is capable of being expressed as an element ofan affinity acceptor tagged HLA-peptide complex.

The affinity tags can be placed on either the N-terminus or C-terminusof the HLA allele. In some embodiments, the affinity tag placed atC-terminus of the HLA allele to enable HLA-peptide localization to cellsurface vs. ER. In some embodiments, the affinity tag placed atN-terminus of the HLA allele to enable single-HLA isolations from celllines expressing multiple endogenous HLA alleles. In yet anotherembodiment, the affinity tag is added to variable β-chains toimmunopurify specific class II HLA heterodimers.

In some embodiments, a cleavage sequence, such as F2A, or an internalribosome entry site (IRES) can be placed between the α-chain andβ2-microglobulin (class I) or between the α-chain and β-chain (classII). In some embodiments, a single class I HLA allele is HLA-A*02:01,HLA-A*23:01 and HLA-B*14:02, or HLA-E*01:01, and class II HLA allele isHLA-DRB*01:01, HLA-DRB*01:02 and HLA-DRB*11:01, HLA-DRB*15:01, orHLA-DRB*07:01.

Non-limiting exemplary affinity acceptor tagged HLA constructs aredepicted in FIG. 2, FIG. 6C, and FIG. 7C.

Therapeutic Methods

Personalized immunotherapy using tumor-specific peptides has beendescribed (Ott et al., Hematol. Oncol. Clin. N. Am. 28 (2014) 559-569).Efficiently choosing which particular peptides to utilize as animmunogen requires the ability to predict which tumor-specific peptideswould efficiently bind to the HLA alleles present in a patient. One ofthe critical barriers to developing curative and tumor-specificimmunotherapy is the identification and selection of highly specific andrestricted tumor antigens to avoid autoimmunity. Tumor neoantigens,which arise as a result of genetic change (e.g., inversions,translocations, deletions, missense mutations, splice site mutations,etc.) within malignant cells, represent the most tumor-specific class ofantigens. Neoantigens have rarely been used in cancer vaccine orimmunogenic compositions due to technical difficulties in identifyingthem, selecting optimized antigens, and producing neoantigens for use ina vaccine or immunogenic composition. These problems can be addressedby: identifying mutations in neoplasias/tumors which are present at theDNA level in tumor but not in matched germline samples from a highproportion of subjects having cancer; analyzing the identified mutationswith one or more peptide-MHC binding prediction algorithms to generate aplurality of neoantigen T cell epitopes that are expressed within theneoplasia/tumor and that bind to a high proportion of patient HLAalleles; and synthesizing the plurality of neoantigenic peptidesselected from the sets of all neoantigen peptides and predicted bindingpeptides for use in a cancer vaccine or immunogenic composition suitablefor treating a high proportion of subjects having cancer (FIG. 18A andFIG. 18B).

For example, translating peptide sequencing information into atherapeutic vaccine can include prediction of mutated peptides that canbind to HLA molecules of a high proportion of individuals. Efficientlychoosing which particular mutations to utilize as immunogen requires theability to predict which mutated peptides would efficiently bind to ahigh proportion of patient's HLA alleles. Recently, neural network basedlearning approaches with validated binding and non-binding peptides haveadvanced the accuracy of prediction algorithms for the major HLA-A and-B alleles. However, even using advanced neural network-based algorithmsto encode HLA-peptide binding rules, several factors limit the power topredict peptides presented on HLA alleles.

For example, translating peptide sequencing information into atherapeutic vaccine can include formulating the drug as a multi-epitopevaccine of long peptides. Targeting as many mutated epitopes aspractically possible takes advantage of the enormous capacity of theimmune system, prevents the opportunity for immunological escape bydown-modulation of an immune targeted gene product, and compensates forthe known inaccuracy of epitope prediction approaches. Syntheticpeptides provide a useful means to prepare multiple immunogensefficiently and to rapidly translate identification of mutant epitopesto an effective vaccine. Peptides can be readily synthesized chemicallyand easily purified utilizing reagents free of contaminating bacteria oranimal substances. The small size allows a clear focus on the mutatedregion of the protein and also reduces irrelevant antigenic competitionfrom other components (unmutated protein or viral vector antigens).

For example, translating peptide sequencing information into atherapeutic vaccine can include a combination with a strong vaccineadjuvant. Effective vaccines can require a strong adjuvant to initiatean immune response. For example, poly-ICLC, an agonist of TLR3 and theRNA helicase-domains of MDA5 and RIG3, has shown several desirableproperties for a vaccine adjuvant. These properties include theinduction of local and systemic activation of immune cells in vivo,production of stimulatory chemokines and cytokines, and stimulation ofantigen-presentation by DCs. Furthermore, poly-ICLC can induce durableCD4+ and CD8+ responses in humans. Importantly, striking similarities inthe upregulation of transcriptional and signal transduction pathwayswere seen in subjects vaccinated with poly-ICLC and in volunteers whohad received the highly effective, replication-competent yellow fevervaccine. Furthermore, >90% of ovarian carcinoma patients immunized withpoly-ICLC in combination with a NYESO-1 peptide vaccine (in addition toMontanide) showed induction of CD4+ and CD8+ T cell, as well as antibodyresponses to the peptide in a recent phase 1 study. At the same time,poly-ICLC has been extensively tested in more than 25 clinical trials todate and exhibited a relatively benign toxicity profile.

In some embodiments, immunogenic peptides can be identified from cellsfrom a subject with a disease or condition. In some embodiments,immunogenic peptides can be specific to a subject with a disease orcondition. In some embodiments, immunogenic peptides can bind to an HLAthat is matched to an HLA haplotype of a subject with a disease orcondition.

In some embodiments, a library of peptides can be expressed in thecells. In some embodiments, the cells comprise the peptides to beidentified or characterized. In some embodiments, the peptides to beidentified or characterized are endogenous peptides. In someembodiments, the peptides are exogenous peptides. For example, thepeptides to be identified or characterized can be expressed from aplurality of sequences encoding a library of peptides.

Prior to disclosure of the instant specification, the majority ofLC-MS/MS studies of the HLA peptidome have used cells expressingmultiple HLA molecules, which requires peptides to be assigned to 1 ofup to 6 class I alleles using pre-existing bioinformatics predictors or“deconvolution” (Bassani-Sternberg and Gfeller, 2016). Thus, peptidesthat do not closely match known motifs could not confidently be reportedas binders to a given HLA allele.

Provided herein are methods of prediction of peptides, such as mutatedpeptides, that can bind to HLA molecules of individuals. In someembodiments, the application provides methods of identifying from agiven set of antigen comprising peptides the most suitable peptides forpreparing an immunogenic composition for a subject, said methodcomprising selecting from set given set of peptides the plurality ofpeptides capable of binding an HLA protein of the subject, wherein saidability to bind an HLA protein is determined by analyzing the sequenceof peptides with a machine which has been trained with peptide sequencedatabases corresponding to the specific HLA-binding peptides for each ofthe HLA-alleles of said subject. Provided herein are methods ofidentifying from a given set of antigen comprising peptides the mostsuitable peptides for preparing an immunogenic composition for asubject, said method comprising selecting from set given set of peptidesthe plurality of peptides determined as capable of binding an HLAprotein of the subject, ability to bind an HLA protein is determined byanalyzing the sequence of peptides with a machine which has been trainedwith a peptide sequence database obtained by carrying out the methodsdescribed herein above. Thus, in some embodiments, the presentdisclosure provides methods of identifying a plurality ofsubject-specific peptides for preparing a subject-specific immunogeniccomposition, wherein the subject has a tumor and the subject-specificpeptides are specific to the subject and the subject's tumor, saidmethod comprising: sequencing of a sample of the subject's tumor and anon-tumor sample of the subject; determining based on the nucleic acidsequencing: non-silent mutations present in the genome of cancer cellsof the subject but not in normal tissue from the subject, and the HLAgenotype of the subject; and selecting from the identified non-silentmutations the plurality of subject-specific peptides, each having adifferent tumor epitope that is an epitope specific to the tumor of thesubject and each being identified as capable of binding an HLA proteinof the subject, as determined by analyzing the sequence of peptidesderived from the non-silent mutations in the methods for predicting HLAbinding described herein.

In some embodiments, disclosed herein, is a method of characterizingHLA-peptide complexes specific to an individual.

In some embodiments, a method of characterizing HLA-peptide complexesspecific to an individual is used to develop an immunotherapeutic in anindividual in need thereof, such as a subject with a condition ordisease.

Provided herein is a method of providing an anti-tumor immunity in amammal comprising administering to the mammal a polynucleic acidcomprising a sequence encoding a peptide identified according to amethod described Provided herein is a method of providing an anti-tumorimmunity in a mammal comprising administering to the mammal an effectiveamount of a peptide with a sequence of a peptide identified according toa method described herein. Provided herein is a method of providing ananti-tumor immunity in a mammal comprising administering to the mammal acell comprising a peptide comprising the sequence of a peptideidentified according to a method described herein. Provided herein is amethod of providing an anti-tumor immunity in a mammal comprisingadministering to the mammal a cell comprising a polynucleic acidcomprising a sequence encoding a peptide comprising the sequence ofpeptide identified according to a method described herein. In someembodiments, the cell presents the peptide as an HLA-peptide complex.

Provided herein is a method of treating a disease or disorder in asubject, the method comprising administering to the subject apolynucleic acid comprising a sequence encoding a peptide identifiedaccording to a method described herein. Provided herein is a method oftreating a disease or disorder in a subject, the method comprisingadministering to the subject an effective amount of a peptide comprisingthe sequence of a peptide identified according to a method describedherein. Provided herein is a method of treating a disease or disorder ina subject, the method comprising administering to the subject a cellcomprising a peptide comprising the sequence of a peptide identifiedaccording to a method described herein. Provided herein is a method oftreating a disease or disorder in a subject, the method comprisingadministering to the subject a cell comprising a polynucleic acidcomprising a sequence encoding a peptide comprising the sequence of apeptide identified according to a method described herein. In someembodiments, wherein the disease or disorder is cancer. In someembodiments, the method further comprises administering an immunecheckpoint inhibitor to the subject.

Disclosed herein, in some embodiments, are methods of developing animmunotherapeutic for an individual in need thereof by characterizingHLA-peptide complexes comprising: a) providing a population of cellsderived from the individual in need thereof wherein one or more cells ofthe population of cells comprise a polynucleic acid comprising asequence encoding an affinity acceptor tagged class I or class II HLAallele, wherein the sequence encoding an affinity acceptor tagged HLAcomprises: i) a sequence encoding a recombinant class I or class II HLAallele operatively linked to ii) a sequence encoding an affinityacceptor peptide; b) expressing the affinity acceptor tagged HLA in atleast one cell of the one or more cells of the population of cells,thereby forming affinity acceptor tagged HLA-peptide complexes in the atleast one cell; c) enriching for the affinity acceptor taggedHLA-peptide complexes; characterizing HLA-peptide complexes specific tothe individual in need thereof; and d) developing the immunotherapeuticbased on an HLA-peptide complex specific to the individual in needthereof; wherein the individual has a disease or condition.

In some embodiments, the immunotherapeutic is a nucleic acid or apeptide therapeutic.

In some embodiments, the method comprises introducing one or morepeptides to the population of cells. In some embodiments, the methodcomprises contacting the population of cells with the one or morepeptides or expressing the one or more peptides in the population ofcells. In some embodiments, the introducing comprises contacting thepopulation of cells with one or more nucleic acids encoding the one ormore peptides.

In some embodiments, the method comprises introducing one or more HLAsspecific for the patient. In some embodiments, the method comprisesintroducing all HLAs specific for the patient. In some embodiments,patient specific HLAs can be introduced as single allele. In someembodiments, multiple patient specific HLAs can be introduced. In someembodiments, the method comprises developing a immunotherapeutic basedon peptides identified in connection with the patient-specific HLAs]. Insome embodiments, the population of cells is derived from the individualin need thereof.

In some embodiments, the method comprises expressing a library ofpeptides in the population of cells, thereby forming a library ofaffinity acceptor tagged HLA-peptide complexes. In some embodiments, themethod comprises contacting to the population of cells a library ofpeptides or a library of sequences encoding peptides, thereby forming alibrary of affinity acceptor tagged HLA-peptide complexes. In someembodiments, the library comprises a library of peptides associated withthe disease or condition. In some embodiments, the disease or conditionis cancer or an infection with an infectious agent or an autoimmunedisease. In some embodiments, the method comprises introducing theinfectious agent or portions thereof into one or more cells of thepopulation of cells. In some embodiments, the method comprisescharacterizing one or more peptides from the HLA-peptide complexesspecific to the individual in need thereof, optionally wherein thepeptides are from one or more target proteins of the infectious agent orthe autoimmune disease. In some embodiments, the method comprisescharacterizing one or more regions of the peptides from the one or moretarget proteins of the infectious agent or autoimmune disease. In someembodiments, the method comprises identifying peptides from theHLA-peptide complexes derived from an infectious agent or an autoimmunedisease.

In some embodiments, the infectious agent is a pathogen. In someembodiments, the pathogen is a virus, bacteria, or a parasite.

In some embodiments, the virus is selected from the group consisting of:BK virus (BKV), Dengue viruses (DENV-1, DENV-2, DENV-3, DENV-4, DENV-5),cytomegalovirus (CMV), Hepatitis B virus (HBV), Hepatitis C virus (HCV),Epstein-Barr virus (EBV), an adenovirus, human immunodeficiency virus(HIV), human T-cell lymphotrophic virus (HTLV-1), an influenza virus,RSV, HPV, rabies, mumps rubella virus, poliovirus, yellow fever,hepatitis A, hepatitis B, Rotavirus, varicella virus, humanpapillomavirus (HPV), smallpox, zoster, and combinations thereof.

In some embodiments, the bacteria is selected from the group consistingof: Klebsiella spp., Tropheryma whipplei, Mycobacterium leprae,Mycobacterium lepromatosis, and Mycobacterium tuberculosis. In someembodiments, the bacteria is selected from the group consisting of:typhoid, pneumococcal, meningococcal, haemophilus B, anthrax, tetanustoxoid, meningococcal group B, bcg, cholera, and combinations thereof.

In some embodiments, the parasite is a helminth or a protozoan. In someembodiments, the parasite is selected from the group consisting of:Leishmania spp. (e.g. L. major, L. infantum, L. braziliensis, L.donovani, L. chagasi, L. mexicana), Plasmodium spp. (e.g. P. falciparum,P. vivax, P. ovale, P. malariae), Trypanosoma cruzi, Ascarislumbricoides, Trichuris trichiura, Necator americanus, and Schistosomaspp. (S. mansoni, S. haematobium, S. japonicum).

In some embodiments, the immunotherapeutic is an engineered receptor. Insome embodiments, the engineered receptor is a chimeric antigen receptor(CAR), a T-cell receptor (TCR), or a B-cell receptor (BCR), an adoptiveT cell therapy (ACT), or a derivative thereof. In other aspects, theengineered receptor is a chimeric antigen receptor (CAR). In someaspects, the CAR is a first generation CAR. In other aspects, the CAR isa second generation CAR. In still other aspects, the CAR is a thirdgeneration CAR.

In some aspects, the CAR comprises an extracellular portion, atransmembrane portion, and an intracellular portion. In some aspects,the intracellular portion comprises at least one T cell co-stimulatorydomain. In some aspects, the T cell co-stimulatory domain is selectedfrom the group consisting of CD27, CD28, TNFRS9 (4-1BB), TNFRSF4 (OX40),TNFRSF8 (CD30), CD40LG (CD40L), ICOS, ITGB2 (LFA-1), CD2, CD7, KLRC2(NKG2C), TNFRS18 (GITR), TNFRSF14 (HVEM), or any combination thereof.

In some aspects, the engineered receptor binds a target. In someaspects, the binding is specific to a peptide identified from the methodof characterizing HLA-peptide complexes specific to an individualsuffering from a disease or condition.

In some aspects, the immunotherapeutic is a cell as described in detailherein. In some aspects, the immunotherapeutic is a cell comprising areceptor that specifically binds a peptide identified from the methodcharacterizing HLA-peptide complexes specific to an individual sufferingfrom a disease or condition. In some aspects, the immunotherapeutic is acell used in combination with the peptides/nucleic acids of thisinvention. In some embodiments, the cell is a patient cell. In someembodiments, the cell is a T cell. In some embodiments, the cell istumor infiltrating lymphocyte.

In some aspects, a subject with a condition or disease is treated basedon a T cell receptor repertoire of the subject. In some embodiments, anantigen vaccine is selected based on a T cell receptor repertoire of thesubject. In some embodiments, a subject is treated with T cellsexpressing TCRs specific to an antigen or peptide identified using themethods described herein. In some embodiments, a subject is treated withan antigen or peptide identified using the methods described hereinspecific to TCRs, e.g., subject specific TCRs. In some embodiments, asubject is treated with an antigen or peptide identified using themethods described herein specific to T cells expressing TCRs, e.g.,subject specific TCRs. In some embodiments, a subject is treated with anantigen or peptide identified using the methods described hereinspecific to subject specific TCRs.

In some embodiments, an immunogenic antigen composition or vaccine isselected based on TCRs identified in a subject. In one embodimentidentification of a T cell repertoire and testing in functional assaysis used to determine an immunogenic composition or vaccine to beadministered to a subject with to condition or disease. In someembodiments, the immunogenic composition is an antigen vaccine. In someembodiments, the antigen vaccine comprises subject specific antigenpeptides. In some embodiments, antigen peptides to be included in anantigen vaccine are selected based on a quantification of subjectspecific TCRs that bind to the antigens. In some embodiments, antigenpeptides are selected based on a binding affinity of the peptide to aTCR. In some embodiments, the selecting is based on a combination ofboth the quantity and the binding affinity. For example, a TCR thatbinds strongly to an antigen in a functional assay, but that is nothighly represented in a TCR repertoire can be a good candidate for anantigen vaccine because T cells expressing the TCR would beadvantageously amplified.

In some embodiments, antigens are selected for administering to asubject based on binding to TCRs. In some embodiments, T cells, such asT cells from a subject with a disease or condition, can be expanded.Expanded T cells that express TCRs specific to an immunogenic antigenpeptide identified using the method described herein, can beadministered back to a subject. In some embodiments, suitable cells,e.g., PBMCs, are transduced or transfected with polynucleotides forexpression of TCRs specific to an immunogenic antigen peptide identifiedusing the method described herein and administered to a subject. T cellsexpressing TCRs specific to an immunogenic antigen peptide identifiedusing the method described herein can be expanded and administered backto a subject. In some embodiments, T cells that express TCRs specific toan immunogenic antigen peptide identified using the method describedherein that result in cytolytic activity when incubated with autologousdiseased tissue can be expanded and administered to a subject. In someembodiments, T cells used in functional assays result in binding to animmunogenic antigen peptide identified using the method described hereincan be expanded and administered to a subject. In some embodiments, TCRsthat have been determined to bind to subject specific immunogenicantigen peptides identified using the method described herein can beexpressed in T cells and administered to a subject.

The methods described herein can involve adoptive transfer of immunesystem cells, such as T cells, specific for selected antigens, such astumor or pathogen associated antigens. Various strategies can beemployed to genetically modify T cells by altering the specificity ofthe T cell receptor (TCR) for example by introducing new TCR α and βchains with specificity to an immunogenic antigen peptide identifiedusing the method described herein (see, e.g., U.S. Pat. No. 8,697,854;PCT Patent Publications: WO2003020763, WO2004033685, WO2004044004,WO2005114215, WO2006000830, WO2008038002, WO2008039818, WO2004074322,WO2005113595, WO2006125962, WO2013166321, WO2013039889, WO2014018863,WO2014083173; U.S. Pat. No. 8,088,379).

Chimeric antigen receptors (CARs) can be used to generateimmunoresponsive cells, such as T cells, specific for selected targets,such a immunogenic antigen peptides identified using the methoddescribed herein, with a wide variety of receptor chimera constructs(see, e.g., U.S. Pat. Nos. 5,843,728; 5,851,828; 5,912, 170; 6,004,811;6,284,240; 6,392,013; 6,410,014; 6,753,162; 8,211,422; and, PCTPublication WO9215322). Alternative CAR constructs can be characterizedas belonging to successive generations. First-generation CARs typicallyconsist of a single-chain variable fragment of an antibody specific foran antigen, for example comprising a VL linked to a VH of a specificantibody, linked by a flexible linker, for example by a CD8a hingedomain and a CD8a transmembrane domain, to the transmembrane andintracellular signaling domains of either CD3ζ or FcRy or scFv-FcRy(see, e.g., U.S. Pat. Nos. 7,741,465; 5,912,172; 5,906,936).Second-generation CARs incorporate the intracellular domains of one ormore costimulatory molecules, such as CD28, OX40 (CD134), or 4-1BB(CD137) within the endodomain, e.g., scFv-CD28/OX40/4-1BB-CD3 (see,e.g., U.S. Pat. Nos. 8,911,993; 8,916,381; 8,975,071; 9,101,584;9,102,760; 9,102,761). Third-generation CARs include a combination ofcostimulatory endodomains, such a CD3C-chain, CD97, GDI 1a-CD18, CD2,ICOS, CD27, CD154, CDS, OX40, 4-1BB, or CD28 signaling domains, e.g.,scFv-CD28-4-1BB-CD3C or scFv-CD28-OX40-CD3Q (see, e.g., U.S. Pat. Nos.8,906,682; 8,399,645; 5,686,281; PCT Publication No. WO2014134165; PCTPublication No. WO2012079000). In some embodiments, costimulation can becoordinated by expressing CARs in antigen-specific T cells, chosen so asto be activated and expanded following, for example, interaction withantigen on professional antigen-presenting cells, with costimulation.Additional engineered receptors can be provided on the immunoresponsivecells, e.g., to improve targeting of a T-cell attack and/or minimizeside effects.

Alternative techniques can be used to transform target immunoresponsivecells, such as protoplast fusion, lipofection, transfection orelectroporation. A wide variety of vectors can be used, such asretroviral vectors, lentiviral vectors, adenoviral vectors,adeno-associated viral vectors, plasmids or transposons, such as aSleeping Beauty transposon (see U.S. Pat. Nos. 6,489,458; 7,148,203;7,160,682; 7,985,739; 8,227,432), can be used to introduce CARs, forexample using 2nd generation antigen-specific CARs signaling throughCD3ζ and either CD28 or CD137. Viral vectors can for example includevectors based on HIV, SV40, EBV, HSV or BPV

Cells that are targeted for transformation can for example include Tcells, Natural Killer (NK) cells, cytotoxic T lymphocytes (CTL),regulatory T cells, human embryonic stem cells, tumor-infiltratinglymphocytes (TIL) or a pluripotent stem cell from which lymphoid cellscan be differentiated. T cells expressing a desired CAR can for examplebe selected through co-culture with γ-irradiated activating andpropagating cells (APC), which co-express the cancer antigen andco-stimulatory molecules. The engineered CAR T-cells can be expanded,for example by co-culture on APC in presence of soluble factors, such asIL-2 and IL-21. This expansion can for example be carried out so as toprovide memory CAR T cells (which for example be assayed bynon-enzymatic digital array and/or multi-panel flow cytometry). In thisway, CAR T cells can be provided that have specific cytotoxic activityagainst antigen-bearing tumors (optionally in conjunction withproduction of desired chemokines such as interferon-γ). CAR T cells ofthis kind can for example be used in animal models, for example tothreat tumor xenografts.

Approaches such as the foregoing can be adapted to provide methods oftreating and/or increasing survival of a subject having a disease, suchas a neoplasia or pathogenic infection, for example by administering aneffective amount of an immunoresponsive cell comprising an antigenrecognizing receptor that binds a selected antigen, wherein the bindingactivates the immunoresponsive cell, thereby treating or preventing thedisease (such as a neoplasia, a pathogen infection, an autoimmunedisorder, or an allogeneic transplant reaction). Dosing in CAR T celltherapies can for example involve administration of from 10⁶ to 10⁹cells/kg, with or without a course of lymphodepletion, for example withcyclophosphamide.

To guard against possible adverse reactions, engineered immunoresponsivecells can be equipped with a transgenic safety switch, in the form of atransgene that renders the cells vulnerable to exposure to a specificsignal. For example, the herpes simplex viral thymidine kinase (TK) genecan be used in this way, for example by introduction into allogeneic Tlymphocytes used as donor lymphocyte infusions following stem celltransplantation. In such cells, administration of a nucleoside prodrugsuch as ganciclovir or acyclovir causes cell death. Alternative safetyswitch constructs include inducible caspase 9, for example triggered byadministration of a small-molecule dimerizer that brings together twononfunctional icasp9 molecules to form the active enzyme. A wide varietyof alternative approaches to implementing cellular proliferationcontrols have been described (see, e.g., U.S. Patent Publication No.20130071414; PCT Patent Publication WO2011146862; PCT Patent PublicationWO201401 1987; PCT Patent Publication WO2013040371). In a furtherrefinement of adoptive therapies, genome editing can be used to tailorimmunoresponsive cells to alternative implementations, for exampleproviding edited CAR T cells.

Cell therapy methods can also involve the ex-vivo activation andexpansion of T-cells. In some embodiments, T cells can be activatedbefore administering them to a subject in need thereof. Examples ofthese type of treatments include the use tumor infiltrating lymphocyte(TIL) cells (see U.S. Pat. No. 5,126,132), cytotoxic T-cells (see U.S.Pat. Nos. 6,255,073; and 5,846,827), expanded tumor draining lymph nodecells (see U.S. Pat. No. 6,251,385), and various other lymphocytepreparations (see U.S. Pat. Nos. 6,194,207; 5,443,983; 6,040,177; and5,766,920).

An ex vivo activated T-cell population can be in a state that maximallyorchestrates an immune response to cancer, infectious diseases, or otherdisease states, e.g., an autoimmune disease state. For activation, atleast two signals can be delivered to the T cells. The first signal isnormally delivered through the T-cell receptor (TCR) on the T-cellsurface. The TCR first signal is normally triggered upon interaction ofthe TCR with peptide antigens expressed in conjunction with an MHCcomplex on the surface of an antigen-presenting cell (APC). The secondsignal is normally delivered through co-stimulatory receptors on thesurface of T-cells. Co-stimulatory receptors are generally triggered bycorresponding ligands or cytokines expressed on the surface of APCs.

It is contemplated that the T cells specific to immunogenic antigenpeptides identified using the method described herein can be obtainedand used in methods of treating or preventing disease. In this regard,the disclosure provides a method of treating or preventing a disease orcondition in a subject, comprising administering to the subject a cellpopulation comprising cells specific to immunogenic antigen peptidesidentified using the method described herein in an amount effective totreat or prevent the disease in the subject. In some embodiments, amethod of treating or preventing a disease in a subject, comprisesadministering a cell population enriched for disease-reactive T cells toa subject in an amount effective to treat or prevent cancer in themammal. The cells can be cells that are allogeneic or autologous to thesubject.

The disclosure further provides a method of inducing a disease specificimmune response in a subject, vaccinating against a disease, treatingand/or alleviating a symptom of a disease in a subject by administeringthe subject an antigenic peptide or vaccine.

The peptide or composition of the disclosure can be administered in anamount sufficient to induce a CTL response. An antigenic peptide orvaccine composition can be administered alone or in combination withother therapeutic agents. Exemplary therapeutic agents include, but arenot limited to, a chemotherapeutic or biotherapeutic agent, radiation,or immunotherapy. Any suitable therapeutic treatment for a particulardisease can be administered. Examples of chemotherapeutic andbiotherapeutic agents include, but are not limited to, aldesleukin,altretamine, amifostine, asparaginase, bleomycin, capecitabine,carboplatin, carmustine, cladribine, cisapride, cisplatin,cyclophosphamide, cytarabine, dacarbazine (DTIC), dactinomycin,docetaxel, doxorubicin, dronabinol, epoetin alpha, etoposide,filgrastim, fludarabine, fluorouracil, gemcitabine, granisetron,hydroxyurea, idarubicin, ifosfamide, interferon alpha, irinotecan,lansoprazole, levamisole, leucovorin, megestrol, mesna, methotrexate,metoclopramide, mitomycin, mitotane, mitoxantrone, omeprazole,ondansetron, paclitaxel (Taxol®), pilocarpine, prochloroperazine,rituximab, tamoxifen, taxol, topotecan hydrochloride, trastuzumab,vinblastine, vincristine and vinorelbine tartrate. In addition, thesubject can be further administered an anti-immunosuppressive orimmunostimulatory agent. For example, the subject can be furtheradministered an anti-CTLA antibody or anti-PD-1 or anti-PD-L1.

The amount of each peptide to be included in a vaccine composition andthe dosing regimen can be determined by one skilled in the art. Forexample, a peptide or its variant can be prepared for intravenous (i.v.)injection, sub-cutaneous (s.c.) injection, intradermal (i.d.) injection,intraperitoneal (i.p.) injection, intramuscular (i.m.) injection.Exemplary methods of peptide injection include s.c, i.d., i.p., i.m.,and i.v. Exemplary methods of DNA injection include i.d., i.m., s.c,i.p. and i.v. Other methods of administration of the vaccine compositionare known to those skilled in the art.

A pharmaceutical composition can be compiled such that the selection,number and/or amount of peptides present in the composition is/aredisease and/or patient-specific. For example, the exact selection ofpeptides can be guided by expression patterns of the parent proteins ina given tissue to avoid side effects. The selection can be dependent onthe specific type of disease, the status of the disease, earliertreatment regimens, the immune status of the patient, and theHLA-haplotype of the patient. Furthermore, the vaccine according to thepresent disclosure can contain individualized components, according topersonal needs of the particular patient. Examples include varying theamounts of peptides according to the expression of the related antigenin the particular patient, unwanted side-effects due to personalallergies or other treatments, and adjustments for secondary treatmentsfollowing a first round or scheme of treatment.

Production of Disease Specific Antigens

The present disclosure is based, at least in part, on the ability topresent the immune system of the patient with one or moredisease-specific antigens. One of skill in the art from this disclosureand the knowledge in the art will appreciate that there are a variety ofways in which to produce such disease specific antigens. In general,such disease specific antigens can be produced either in vitro or invivo. Disease specific antigens can be produced in vitro as peptides orpolypeptides, which can then be formulated into a vaccine or immunogeniccomposition and administered to a subject. As described in furtherdetail herein, such in vitro production can occur by a variety ofmethods known to one of skill in the art such as, for example, peptidesynthesis or expression of a peptide/polypeptide from a DNA or RNAmolecule in any of a variety of bacterial, eukaryotic, or viralrecombinant expression systems, followed by purification of theexpressed peptide/polypeptide. Alternatively, disease specific antigenscan be produced in vivo by introducing molecules (e.g., DNA, RNA, viralexpression systems, and the like) that encode disease specific antigensinto a subject, whereupon the encoded disease specific antigens areexpressed. The methods of in vitro and in vivo production of antigens isalso further described herein as it relates to pharmaceuticalcompositions and methods of delivery of the therapy.

In some embodiments, the present disclosure includes modified antigenicpeptides. A modification can include a covalent chemical modificationthat does not alter the primary amino acid sequence of the antigenicpeptide itself. Modifications can produce peptides with desiredproperties, for example, prolonging the in vivo half-life, increasingthe stability, reducing the clearance, altering the immunogenicity orallergenicity, enabling the raising of particular antibodies, cellulartargeting, antigen uptake, antigen processing, MHC affinity, MHCstability, or antigen presentation. Changes to an antigenic peptide thatcan be carried out include, but are not limited to, conjugation to acarrier protein, conjugation to a ligand, conjugation to an antibody,PEGylation, polysialylation HESylation, recombinant PEG mimetics, Fcfusion, albumin fusion, nanoparticle attachment, nanoparticulateencapsulation, cholesterol fusion, iron fusion, acylation, amidation,glycosylation, side chain oxidation, phosphorylation, biotinylation, theaddition of a surface active material, the addition of amino acidmimetics, or the addition of unnatural amino acids.

Issues associated with short plasma half-life or susceptibility toprotease degradation can be overcome by various modifications, includingconjugating or linking the polypeptide sequence to any of a variety ofnon-proteinaceous polymers, e.g., polyethylene glycol (PEG),polypropylene glycol, or polyoxyalkylenes (see, for example, typicallyvia a linking moiety covalently bound to both the protein and thenonproteinaceous polymer, e.g., a PEG). Such PEG conjugated biomoleculeshave been shown to possess clinically useful properties, includingbetter physical and thermal stability, protection against susceptibilityto enzymatic degradation, increased solubility, longer in vivocirculating half-life and decreased clearance, reduced immunogenicityand antigenicity, and reduced toxicity.

PEGs suitable for conjugation to a polypeptide sequence are generallysoluble in water at room temperature, and have the general formulaR(O—CH₂—CH₂)nO—R, where R is hydrogen or a protective group such as analkyl or an alkanol group, and where n is an integer from 1 to 1000.When R is a protective group, it generally has from 1 to 8 carbons. ThePEG conjugated to the polypeptide sequence can be linear or branched.Branched PEG derivatives, “star-PEGs” and multi-armed PEGs arecontemplated by the present disclosure.

The present disclosure also contemplates compositions of conjugateswherein the PEGs have different n values and thus the various differentPEGs are present in specific ratios. For example, some compositionscomprise a mixture of conjugates where n=1, 2, 3 and 4. In somecompositions, the percentage of conjugates where n=1 is 18-25%, thepercentage of conjugates where n=2 is 50-66%, the percentage ofconjugates where n=3 is 12-16%, and the percentage of conjugates wheren=4 is up to 5%. Such compositions can be produced by reactionconditions and purification methods know in the art. For example, cationexchange chromatography can be used to separate conjugates, and afraction is then identified which contains the conjugate having, forexample, the desired number of PEGs attached, purified free fromunmodified protein sequences and from conjugates having other numbers ofPEGs attached.

PEG can be bound to a polypeptide of the present disclosure via aterminal reactive group (a “spacer”). The spacer is, for example, aterminal reactive group which mediates a bond between the free amino orcarboxyl groups of one or more of the polypeptide sequences andpolyethylene glycol. The PEG having the spacer which can be bound to thefree amino group includes N-hydroxysuccinylimide polyethylene glycolwhich can be prepared by activating succinic acid ester of polyethyleneglycol with N-hydroxy succinylimide. Another activated polyethyleneglycol which can be bound to a free amino group is2,4-bis(O-methoxypolyethyleneglycol)-6-chloro-s-triazine which can beprepared by reacting polyethylene glycol monomethyl ether with cyanuricchloride. The activated polyethylene glycol which is bound to the freecarboxyl group includes polyoxyethylenediamine.

Conjugation of one or more of the polypeptide sequences of the presentdisclosure to PEG having a spacer can be carried out by variousconventional methods. For example, the conjugation reaction can becarried out in solution at a pH of from 5 to 10, at temperature from 4°C. to room temperature, for 30 minutes to 20 hours, utilizing a molarratio of reagent to protein of from 4:1 to 30:1. Reaction conditions canbe selected to direct the reaction towards producing predominantly adesired degree of substitution. In general, low temperature, low pH(e.g., pH=5), and short reaction time tend to decrease the number ofPEGs attached, whereas high temperature, neutral to high pH (e.g.,pH>7), and longer reaction time tend to increase the number of PEGsattached. Various means known in the art can be used to terminate thereaction. In some embodiments the reaction is terminated by acidifyingthe reaction mixture and freezing at, e.g., −20° C.

The present disclosure also contemplates the use of PEG mimetics.Recombinant PEG mimetics have been developed that retain the attributesof PEG (e.g., enhanced serum half-life) while conferring severaladditional advantageous properties. By way of example, simplepolypeptide chains (comprising, for example, Ala, Glu, Gly, Pro, Ser andThr) capable of forming an extended conformation similar to PEG can beproduced recombinantly already fused to the peptide or protein drug ofinterest (e.g., Amunix's XTEN technology; Mountain View, Calif.). Thisobviates the need for an additional conjugation step during themanufacturing process. Moreover, established molecular biologytechniques enable control of the side chain composition of thepolypeptide chains, allowing optimization of immunogenicity andmanufacturing properties.

Glycosylation can affect the physical properties of proteins and canalso be important in protein stability, secretion, and subcellularlocalization. Proper glycosylation can be important for biologicalactivity. In fact, some genes from eukaryotic organisms, when expressedin bacteria (e.g., E. coli) which lack cellular processes forglycosylating proteins, yield proteins that are recovered with little orno activity by virtue of their lack of glycosylation. Addition ofglycosylation sites can be accomplished by altering the amino acidsequence. The alteration to the polypeptide can be made, for example, bythe addition of, or substitution by, one or more serine or threonineresidues (for O-linked glycosylation sites) or asparagine residues (forN-linked glycosylation sites). The structures of N-linked and O-linkedoligosaccharides and the sugar residues found in each type can bedifferent. One type of sugar that is commonly found on both isN-acetylneuraminic acid (hereafter referred to as sialic acid). Sialicacid is usually the terminal residue of both N-linked and O-linkedoligosaccharides and, by virtue of its negative charge, may conferacidic properties to the glycoprotein. Embodiments of the presentdisclosure comprise the generation and use of N-glycosylation variants.

The polypeptide sequences of the present disclosure can optionally bealtered through changes at the DNA level, particularly by mutating theDNA encoding the polypeptide at preselected bases such that codons aregenerated that will translate into the desired amino acids. Anothermeans of increasing the number of carbohydrate moieties on thepolypeptide is by chemical or enzymatic coupling of glycosides to thepolypeptide. Removal of carbohydrates can be accomplished chemically orenzymatically, or by substitution of codons encoding amino acid residuesthat are glycosylated. Chemical deglycosylation techniques are known,and enzymatic cleavage of carbohydrate moieties on polypeptides can beachieved by the use of a variety of endo- and exo-glycosidases.

Additional suitable components and molecules for conjugation include,for example, molecules for targeting to the lymphatic system,thyroglobulin; albumins such as human serum albumin (HAS); tetanustoxoid; Diphtheria toxoid; polyamino acids such aspoly(D-lysine:D-glutamic acid); VP6 polypeptides of rotaviruses;influenza virus hemagglutinin, influenza virus nucleoprotein; KeyholeLimpet Hemocyanin (KLH); and hepatitis B virus core protein and surfaceantigen; or any combination of the foregoing.

Fusion of albumin to one or more polypeptides of the present disclosurecan, for example, be achieved by genetic manipulation, such that the DNAcoding for HSA, or a fragment thereof, is joined to the DNA coding forthe one or more polypeptide sequences. Thereafter, a suitable host canbe transformed or transfected with the fused nucleotide sequences in theform of, for example, a suitable plasmid, so as to express a fusionpolypeptide. The expression can be effected in vitro from, for example,prokaryotic or eukaryotic cells, or in vivo from, for example, atransgenic organism. In some embodiments of the present disclosure, theexpression of the fusion protein is performed in mammalian cell lines,for example, CHO cell lines. Transformation is used broadly herein torefer to the genetic alteration of a cell resulting from the directuptake, incorporation and expression of exogenous genetic material(exogenous DNA) from its surroundings and taken up through the cellmembrane(s). Transformation occurs naturally in some species ofbacteria, but it can also be effected by artificial means in othercells. Furthermore, albumin itself can be modified to extend itscirculating half-life. Fusion of the modified albumin to one or morepolypeptides can be attained by the genetic manipulation techniquesdescribed above or by chemical conjugation; the resulting fusionmolecule has a half-life that exceeds that of fusions with non-modifiedalbumin. (See WO2011/051489). Several albumin-binding strategies havebeen developed as alternatives for direct fusion, including albuminbinding through a conjugated fatty acid chain (acylation). Because serumalbumin is a transport protein for fatty acids, these natural ligandswith albumin-binding activity have been used for half-life extension ofsmall protein therapeutics. For example, insulin detemir (LEVEMIR), anapproved product for diabetes, comprises a myristyl chain conjugated toa genetically-modified insulin, resulting in a long-acting insulinanalog.

Another type of modification is to conjugate (e.g., link) one or moreadditional components or molecules at the N- and/or C-terminus of apolypeptide sequence, such as another protein (e.g., a protein having anamino acid sequence heterologous to the subject protein), or a carriermolecule. Thus, an exemplary polypeptide sequence can be provided as aconjugate with another component or molecule.

A conjugate modification can result in a polypeptide sequence thatretains activity with an additional or complementary function oractivity of the second molecule. For example, a polypeptide sequence canbe conjugated to a molecule, e.g., to facilitate solubility, storage, invivo or shelf half-life or stability, reduction in immunogenicity,delayed or controlled release in vivo, etc. Other functions oractivities include a conjugate that reduces toxicity relative to anunconjugated polypeptide sequence, a conjugate that targets a type ofcell or organ more efficiently than an unconjugated polypeptidesequence, or a drug to further counter the causes or effects associatedwith a disorder or disease as set forth herein (e.g., diabetes).

A polypeptide can also be conjugated to large, slowly metabolizedmacromolecules such as proteins; polysaccharides, such as sepharose,agarose, cellulose, cellulose beads; polymeric amino acids such aspolyglutamic acid, polylysine; amino acid copolymers; inactivated virusparticles; inactivated bacterial toxins such as toxoid from diphtheria,tetanus, cholera, leukotoxin molecules; inactivated bacteria; anddendritic cells.

Additional candidate components and molecules for conjugation includethose suitable for isolation or purification. Particular non-limitingexamples include binding molecules, such as biotin (biotin-avidinspecific binding pair), an antibody, a receptor, a ligand, a lectin, ormolecules that comprise a solid support, including, for example, plasticor polystyrene beads, plates or beads, magnetic beads, test strips, andmembranes. Purification methods such as cation exchange chromatographycan be used to separate conjugates by charge difference, whicheffectively separates conjugates into their various molecular weights.The content of the fractions obtained by cation exchange chromatographycan be identified by molecular weight using conventional methods, forexample, mass spectroscopy, SDS-PAGE, or other known methods forseparating molecular entities by molecular weight.

In some embodiments, the amino- or carboxyl-terminus of a polypeptidesequence of the present disclosure can be fused with an immunoglobulinFc region (e.g., human Fc) to form a fusion conjugate (or fusionmolecule). Fc fusion conjugates have been shown to increase the systemichalf-life of biopharmaceuticals, and thus the biopharmaceutical productcan require less frequent administration.

Fc binds to the neonatal Fc receptor (FcRn) in endothelial cells thatline the blood vessels, and, upon binding, the Fc fusion molecule isprotected from degradation and re-released into the circulation, keepingthe molecule in circulation longer. This Fc binding is believed to bethe mechanism by which endogenous IgG retains its long plasma half-life.More recent Fc-fusion technology links a single copy of abiopharmaceutical to the Fc region of an antibody to optimize thepharmacokinetic and pharmacodynamic properties of the biopharmaceuticalas compared to traditional Fc-fusion conjugates.

The present disclosure contemplates the use of other modifications,currently known or developed in the future, of the polypeptides toimprove one or more properties. One such method for prolonging thecirculation half-life, increasing the stability, reducing the clearance,or altering the immunogenicity or allergenicity of a polypeptide of thepresent disclosure involves modification of the polypeptide sequences byhesylation, which utilizes hydroxyethyl starch derivatives linked toother molecules in order to modify the molecule's characteristics.Various aspects of hesylation are described in, for example, U.S. PatentAppln. Nos. 2007/0134197 and 2006/0258607.

In Vitro Peptide/Polypeptide Synthesis

Proteins or peptides can be made by any technique known to those ofskill in the art, including the expression of proteins, polypeptides orpeptides through standard molecular biological techniques, the isolationof proteins or peptides from natural sources, in vitro translation, orthe chemical synthesis of proteins or peptides.

Peptides can be readily synthesized chemically utilizing reagents thatare free of contaminating bacterial or animal substances (Merrifield RB: Solid phase peptide synthesis. I. The synthesis of a tetrapeptide. J.Am. Chem. Soc. 85:2149-54, 1963). In some embodiments, antigenicpeptides are prepared by (1) parallel solid-phase synthesis onmulti-channel instruments using uniform synthesis and cleavageconditions; (2) purification over a RP-HPLC column with columnstripping; and re-washing, but not replacement, between peptides;followed by (3) analysis with a limited set of the most informativeassays. The Good Manufacturing Practices (GMP) footprint can be definedaround the set of peptides for an individual patient, thus requiringsuite changeover procedures only between syntheses of peptides fordifferent patients.

Alternatively, a nucleic acid (e.g., a polynucleotide) encoding anantigenic peptide of the present disclosure can be used to produce theantigenic peptide in vitro. The polynucleotide can be, e.g., DNA, cDNA,PNA, CNA, RNA, either single- and/or double-stranded, or native orstabilized forms of polynucleotides, such as e.g. polynucleotides with aphosphorothiate backbone, or combinations thereof and it can containintrons so long as it codes for the peptide. In one embodiment in vitrotranslation is used to produce the peptide. Many exemplary systems existthat one skilled in the art could utilize (e.g., Retic Lysate IVT Kit,Life Technologies, Waltham, Mass.). An expression vector capable ofexpressing a polypeptide can also be prepared. Expression vectors fordifferent cell types are well known in the art and can be selectedwithout undue experimentation. Generally, the DNA is inserted into anexpression vector, such as a plasmid, in proper orientation and correctreading frame for expression. If necessary, the DNA can be linked to theappropriate transcriptional and translational regulatory controlnucleotide sequences recognized by the desired host (e.g., bacteria),although such controls are generally available in the expression vector.The vector is then introduced into the host bacteria for cloning usingstandard techniques (see, e.g., Sambrook et al. (1989) MolecularCloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y.).

Expression vectors comprising the isolated polynucleotides, as well ashost cells containing the expression vectors, are also contemplated. Theantigenic peptides can be provided in the form of RNA or cDNA moleculesencoding the desired antigenic peptides. One or more antigenic peptidesof the disclosure can be encoded by a single expression vector.

In some embodiments, the polynucleotides can comprise the codingsequence for the disease specific antigenic peptide fused in the samereading frame to a polynucleotide which aids, for example, in expressionand/or secretion of a polypeptide from a host cell (e.g., a leadersequence which functions as a secretory sequence for controllingtransport of a polypeptide from the cell). The polypeptide having aleader sequence is a preprotein and can have the leader sequence cleavedby the host cell to form the mature form of the polypeptide.

In some embodiments, the polynucleotides can comprise the codingsequence for the disease specific antigenic peptide fused in the samereading frame to a marker sequence that allows, for example, forpurification of the encoded polypeptide, which can then be incorporatedinto a personalized disease vaccine or immunogenic composition. Forexample, the marker sequence can be a hexa-histidine tag supplied by apQE-9 vector to provide for purification of the mature polypeptide fusedto the marker in the case of a bacterial host, or the marker sequencecan be a hemagglutinin (HA) tag derived from the influenza hemagglutininprotein when a mammalian host (e.g., COS-7 cells) is used. Additionaltags include, but are not limited to, Calmodulin tags, FLAG tags, Myctags, S tags, SBP tags, Softag 1, Softag 3, V5 tag, Xpress tag,Isopeptag, SpyTag, Biotin Carboxyl Carrier Protein (BCCP) tags, GSTtags, fluorescent protein tags (e.g., green fluorescent protein tags),maltose binding protein tags, Nus tags, Strep-tag, thioredoxin tag, TCtag, Ty tag, and the like.

In some embodiments, the polynucleotides can comprise the codingsequence for one or more of the disease specific antigenic peptidesfused in the same reading frame to create a single concatamerizedantigenic peptide construct capable of producing multiple antigenicpeptides.

In some embodiments, isolated nucleic acid molecules having a nucleotidesequence at least 60% identical, at least 65% identical, at least 70%identical, at least 75% identical, at least 80% identical, at least 85%identical, at least 90% identical, at least 95% identical, or at least96%, 97%, 98% or 99% identical to a polynucleotide encoding a diseasespecific antigenic peptide of the present disclosure, can be provided.

The isolated disease specific antigenic peptides described herein can beproduced in vitro (e.g., in the laboratory) by any suitable method knownin the art. Such methods range from direct protein synthetic methods toconstructing a DNA sequence encoding isolated polypeptide sequences andexpressing those sequences in a suitable transformed host. In someembodiments, a DNA sequence is constructed using recombinant technologyby isolating or synthesizing a DNA sequence encoding a wild-type proteinof interest. Optionally, the sequence can be mutagenized bysite-specific mutagenesis to provide functional analogs thereof. See,e.g. Zoeller et al., Proc. Nat'l. Acad. Sci. USA 81:5662-5066 (1984) andU.S. Pat. No. 4,588,585.

In some embodiments, a DNA sequence encoding a polypeptide of interestwould be constructed by chemical synthesis using an oligonucleotidesynthesizer. Such oligonucleotides can be designed based on the aminoacid sequence of the desired polypeptide and selecting those codons thatare favored in the host cell in which the recombinant polypeptide ofinterest is produced. Standard methods can be applied to synthesize anisolated polynucleotide sequence encoding an isolated polypeptide ofinterest. For example, a complete amino acid sequence can be used toconstruct a back-translated gene. Further, a DNA oligomer containing anucleotide sequence coding for the particular isolated polypeptide canbe synthesized. For example, several small oligonucleotides coding forportions of the desired polypeptide can be synthesized and then ligated.The individual oligonucleotides typically contain 5′ or 3′ overhangs forcomplementary assembly

Once assembled (e.g., by synthesis, site-directed mutagenesis, oranother method), the polynucleotide sequences encoding a particularisolated polypeptide of interest is inserted into an expression vectorand optionally operatively linked to an expression control sequenceappropriate for expression of the protein in a desired host. Properassembly can be confirmed by nucleotide sequencing, restriction mapping,and expression of a biologically active polypeptide in a suitable host.As well known in the art, in order to obtain high expression levels of atransfected gene in a host, the gene can be operatively linked totranscriptional and translational expression control sequences that arefunctional in the chosen expression host.

Recombinant expression vectors can be used to amplify and express DNAencoding the disease specific antigenic peptides. Recombinant expressionvectors are replicable DNA constructs which have synthetic orcDNA-derived DNA fragments encoding a disease specific antigenic peptideor a bioequivalent analog operatively linked to suitable transcriptionalor translational regulatory elements derived from mammalian, microbial,viral or insect genes. A transcriptional unit generally comprises anassembly of (1) a genetic element or elements having a regulatory rolein gene expression, for example, transcriptional promoters or enhancers,(2) a structural or coding sequence which is transcribed into mRNA andtranslated into protein, and (3) appropriate transcription andtranslation initiation and termination sequences, as described in detailherein. Such regulatory elements can include an operator sequence tocontrol transcription. The ability to replicate in a host, usuallyconferred by an origin of replication, and a selection gene tofacilitate recognition of transformants can additionally beincorporated. DNA regions are operatively linked when they arefunctionally related to each other. For example, DNA for a signalpeptide (secretory leader) is operatively linked to DNA for apolypeptide if it is expressed as a precursor which participates in thesecretion of the polypeptide; a promoter is operatively linked to acoding sequence if it controls the transcription of the sequence; or aribosome binding site is operatively linked to a coding sequence if itis positioned so as to permit translation. Generally, operatively linkedmeans contiguous, and in the case of secretory leaders, means contiguousand in reading frame. Structural elements intended for use in yeastexpression systems include a leader sequence enabling extracellularsecretion of translated protein by a host cell. Alternatively, whererecombinant protein is expressed without a leader or transport sequence,it can include an N-terminal methionine residue. This residue canoptionally be subsequently cleaved from the expressed recombinantprotein to provide a final product.

Useful expression vectors for eukaryotic hosts, especially mammals orhumans include, for example, vectors comprising expression controlsequences from SV40, bovine papilloma virus, adenovirus andcytomegalovirus. Useful expression vectors for bacterial hosts includeknown bacterial plasmids, such as plasmids from Escherichia coli,including pCR 1, pBR322, pMB9 and their derivatives, wider host rangeplasmids, such as M13 and filamentous single-stranded DNA phages.

Suitable host cells for expression of a polypeptide include prokaryotes,yeast, insect or higher eukaryotic cells under the control ofappropriate promoters. Prokaryotes include gram negative or grampositive organisms, for example E. coli or bacilli. Higher eukaryoticcells include established cell lines of mammalian origin. Cell-freetranslation systems could also be employed. Appropriate cloning andexpression vectors for use with bacterial, fungal, yeast, and mammaliancellular hosts are well known in the art (see Pouwels et al., CloningVectors: A Laboratory Manual, Elsevier, N.Y., 1985).

Various mammalian or insect cell culture systems are also advantageouslyemployed to express recombinant protein. Expression of recombinantproteins in mammalian cells can be performed because such proteins aregenerally correctly folded, appropriately modified and completelyfunctional. Examples of suitable mammalian host cell lines include theCOS-7 lines of monkey kidney cells, described by Gluzman (Cell 23:175,1981), and other cell lines capable of expressing an appropriate vectorincluding, for example, L cells, C127, 3T3, Chinese hamster ovary (CHO),293, HeLa and BHK cell lines. Mammalian expression vectors can comprisenontranscribed elements such as an origin of replication, a suitablepromoter and enhancer linked to the gene to be expressed, and other 5′or 3′ flanking nontranscribed sequences, and 5′ or 3′ nontranslatedsequences, such as necessary ribosome binding sites, a polyadenylationsite, splice donor and acceptor sites, and transcriptional terminationsequences. Baculovirus systems for production of heterologous proteinsin insect cells are reviewed by Luckow and Summers, Bio/Technology 6:47(1988).

The proteins produced by a transformed host can be purified according toany suitable method. Such standard methods include chromatography (e.g.,ion exchange, affinity and sizing column chromatography, and the like),centrifugation, differential solubility, or by any other standardtechnique for protein purification. Affinity tags such as hexahistidine,maltose binding domain, influenza coat sequence,glutathione-S-transferase, and the like can be attached to the proteinto allow easy purification by passage over an appropriate affinitycolumn. Isolated proteins can also be physically characterized usingsuch techniques as proteolysis, nuclear magnetic resonance and x-raycrystallography. For example, supernatants from systems which secreterecombinant protein into culture media can be first concentrated using acommercially available protein concentration filter, for example, anAmicon or Millipore Pellicon ultrafiltration unit. Following theconcentration step, the concentrate can be applied to a suitablepurification matrix. Alternatively, an anion exchange resin can beemployed, for example, a matrix or substrate having pendantdiethylaminoethyl (DEAE) groups. The matrices can be acrylamide,agarose, dextran, cellulose or other types commonly employed in proteinpurification. Alternatively, a cation exchange step can be employed.Suitable cation exchangers include various insoluble matrices comprisingsulfopropyl or carboxymethyl groups. Finally, one or more reversed-phasehigh performance liquid chromatography (RP-HPLC) steps employinghydrophobic RP-HPLC media, e.g., silica gel having pendant methyl orother aliphatic groups, can be employed to further purify a cancer stemcell protein-Fc composition. Some or all of the foregoing purificationsteps, in various combinations, can also be employed to provide ahomogeneous recombinant protein.

Recombinant protein produced in bacterial culture can be isolated, forexample, by initial extraction from cell pellets, followed by one ormore concentration, salting-out, aqueous ion exchange or size exclusionchromatography steps. High performance liquid chromatography (HPLC) canbe employed for final purification steps. Microbial cells employed inexpression of a recombinant protein can be disrupted by any convenientmethod, including freeze-thaw cycling, sonication, mechanicaldisruption, or use of cell lysing agents.

In Vivo Peptide/Polypeptide Synthesis

The present disclosure also contemplates the use of nucleic acidmolecules as vehicles for delivering antigenic peptides/polypeptides tothe subject in need thereof, in vivo, in the form of, e.g., DNA/RNAvaccines (see, e.g., WO2012/159643, and WO2012/159754, herebyincorporated by reference in their entirety).

In some embodiments, antigens can be administered to a patient in needthereof by use of a plasmid. These are plasmids which usually consist ofa strong viral promoter to drive the in vivo transcription andtranslation of the gene (or complementary DNA) of interest (Mor, et al.,(1995). The Journal of Immunology 155 (4): 2039-2046). Intron A cansometimes be included to improve mRNA stability and hence increaseprotein expression (Leitner, et al. (1997). The Journal of Immunology159 (12): 6112-6119). Plasmids also include a strongpolyadenylation/transcriptional termination signal, such as bovinegrowth hormone or rabbit beta-globulin polyadenylation sequences(Alarcon et al., (1999). Adv. Parasitol. Advances in Parasitology 42:343-410; Robinson et al., (2000). Adv. Virus Res. Advances in VirusResearch 55: 1-74; Böhmet al., (1996). Journal of Immunological Methods193 (1): 29-40.). Multicistronic vectors are sometimes constructed toexpress more than one immunogen, or to express an immunogen and animmunostimulatory protein (Lewis et al., (1999). Advances in VirusResearch (Academic Press) 54: 129-88)

Plasmids can be introduced into animal tissues by a number of differentmethods. The two most popular approaches are injection of DNA in saline,using a standard hypodermic needle, and gene gun delivery. A schematicoutline of the construction of a DNA vaccine plasmid and its subsequentdelivery by these two methods into a host is illustrated at ScientificAmerican (Weiner et al., (1999) Scientific American 281 (1): 34-41).Injection in saline is normally conducted intramuscularly (IM) inskeletal muscle, or intradermally (ID), with DNA being delivered to theextracellular spaces. This can be assisted by electroporation bytemporarily damaging muscle fibers with myotoxins such as bupivacaine;or by using hypertonic solutions of saline or sucrose (Alarcon et al.,(1999). Adv. Parasitol. Advances in Parasitology 42: 343-410). Immuneresponses to this method of delivery can be affected by many factors,including needle type, needle alignment, speed of injection, volume ofinjection, muscle type, and age, sex and physiological condition of theanimal being injected (Alarcon et al., (1999). Adv. Parasitol. Advancesin Parasitology 42: 343-410).

Gene gun delivery, the other commonly used method of delivery,ballistically accelerates plasmid DNA (pDNA) that has been adsorbed ontogold or tungsten microparticles into the target cells, using compressedhelium as an accelerant (Alarcon et al., (1999). Adv. Parasitol.Advances in Parasitology 42: 343-410; Lewis et al., (1999). Advances inVirus Research (Academic Press) 54: 129-88).

Alternative delivery methods can include aerosol instillation of nakedDNA on mucosal surfaces, such as the nasal and lung mucosa, (Lewis etal., (1999). Advances in Virus Research (Academic Press) 54: 129-88) andtopical administration of pDNA to the eye and vaginal mucosa (Lewis etal., (1999) Advances in Virus Research (Academic Press) 54: 129-88).Mucosal surface delivery has also been achieved using cationicliposome-DNA preparations, biodegradable microspheres, attenuatedShigella or Listeria vectors for oral administration to the intestinalmucosa, and recombinant adenovirus vectors. DNA or RNA can also bedelivered to cells following mild mechanical disruption of the cellmembrane, temporarily permeabilizing the cells. Such a mild mechanicaldisruption of the membrane can be accomplished by gently forcing cellsthrough a small aperture (Ex vivo Cytosolic Delivery of FunctionalMacromolecules to Immune Cells, Sharei et al, PLOS ONE |DOI:10.1371/journal.pone.0118803 Apr. 13, 2015).

In some embodiments, a disease specific vaccine or immunogeniccomposition can include separate DNA plasmids encoding, for example, oneor more antigenic peptides/polypeptides as identified in according tothe disclosure. As discussed herein, the exact choice of expressionvectors can depend upon the peptide/polypeptides to be expressed, and iswell within the skill of the ordinary artisan. The expected persistenceof the DNA constructs (e.g., in an episomal, non-replicating,non-integrated form in the muscle cells) is expected to provide anincreased duration of protection.

One or more antigenic peptides of the present disclosure can be encodedand expressed in vivo using a viral based system (e.g., an adenovirussystem, an adeno associated virus (AAV) vector, a poxvirus, or alentivirus). In one embodiment, the disease vaccine or immunogeniccomposition can include a viral based vector for use in a human patientin need thereof, such as, for example, an adenovirus (see, e.g., Badenet al. First-in-human evaluation of the safety and immunogenicity of arecombinant adenovirus serotype 26 HIV-1 Env vaccine (IPCAVD 001). JInfect Dis. 2013 Jan. 15; 207(2):240-7, hereby incorporated by referencein its entirety). Plasmids that can be used for adeno associated virus,adenovirus, and lentivirus delivery have been described previously (seee.g., U.S. Pat. Nos. 6,955,808 and 6,943,019, and U.S. Patentapplication No. 20080254008, hereby incorporated by reference).

The peptides and polypeptides of the disclosure can also be expressed bya vector, e.g., a nucleic acid molecule as herein-discussed, e.g., RNAor a DNA plasmid, a viral vector such as a poxvirus, e.g., orthopoxvirus, avipox virus, or adenovirus, AAV or lentivirus. This approachinvolves the use of a vector to express nucleotide sequences that encodethe peptide of the disclosure. Upon introduction into an acutely orchronically infected host or into a noninfected host, the vectorexpresses the immunogenic peptide, and thereby elicits a host CTLresponse.

Among vectors that can be used in the practice of the disclosure,integration in the host genome of a cell is possible with retrovirusgene transfer methods, often resulting in long term expression of theinserted transgene. In some embodiments, the retrovirus is a lentivirus.Additionally, high transduction efficiencies have been observed in manydifferent cell types and target tissues. The tropism of a retrovirus canbe altered by incorporating foreign envelope proteins, expanding thepotential target population of target cells. A retrovirus can also beengineered to allow for conditional expression of the insertedtransgene, such that only certain cell types are infected by thelentivirus. Cell type specific promoters can be used to targetexpression in specific cell types. Lentiviral vectors are retroviralvectors (and hence both lentiviral and retroviral vectors can be used inthe practice of the disclosure). Moreover, lentiviral vectors are ableto transduce or infect non-dividing cells and typically produce highviral titers. Selection of a retroviral gene transfer system cantherefore depend on the target tissue. Retroviral vectors are comprisedof cis-acting long terminal repeats with packaging capacity for up to6-10 kb of foreign sequence. The minimum cis-acting LTRs are sufficientfor replication and packaging of the vectors, which are then used tointegrate the desired nucleic acid into the target cell to providepermanent expression. Widely used retroviral vectors that can be used inthe practice of the disclosure include those based upon murine leukemiavirus (MuLV), gibbon ape leukemia virus (GaLV), Simian Immunodeficiencyvirus (SIV), human immunodeficiency virus (HIV), and combinationsthereof (see, e.g., Buchscher et al., (1992) J. Virol. 66:2731-2739;Johann et al., (1992) J. Virol. 66:1635-1640; Sommnerfelt et al., (1990)Virol. 176:58-59; Wilson et al., (1998) J. Virol. 63:2374-2378; Milleret al., (1991) J. Virol. 65:2220-2224; PCT/US94/05700).

Also useful in the practice of the disclosure is a minimal non-primatelentiviral vector, such as a lentiviral vector based on the equineinfectious anemia virus (EIAV) (see, e.g., Balagaan, (2006) J Gene Med;8: 275-285, Published online 21 Nov. 2005 in Wiley InterScience(www.interscience.wiley.com). DOI: 10.1002/jgm.845). The vectors canhave cytomegalovirus (CMV) promoter driving expression of the targetgene. Accordingly, the disclosure contemplates amongst vector(s) usefulin the practice of the disclosure: viral vectors, including retroviralvectors and lentiviral vectors.

Lentiviral vectors have been disclosed as in the treatment forParkinson's Disease, see, e.g., US Patent Publication No. 20120295960and U.S. Pat. Nos. 7,303,910 and 7,351,585. Lentiviral vectors have alsobeen disclosed for delivery to the Brain, see, e.g., US PatentPublication Nos. US20110293571; US20040013648, US20070025970,US20090111106 and U.S. Pat. No. 7,259,015. In another embodimentlentiviral vectors are used to deliver vectors to the brain of thosebeing treated for a disease. As to lentivirus vector systems useful inthe practice of the disclosure, mention is made of U.S. Pat. Nos.6,428,953, 6,165,782, 6,013,516, 5,994,136, 6,312,682, and 7,198,784,and documents cited therein. In an embodiment herein the delivery is viaan lentivirus. Zou et al. administered about 10 μL of a recombinantlentivirus having a titer of 1×10⁹ transducing units (TU)/ml by anintrathecal catheter. These sort of dosages can be adapted orextrapolated to use of a retroviral or lentiviral vector in the presentdisclosure. For transduction in tissues such as the brain, it isnecessary to use very small volumes, so the viral preparation isconcentrated by ultracentrifugation. Other methods of concentration suchas ultrafiltration or binding to and elution from a matrix can be used.In other embodiments the amount of lentivirus administered can be 1×10⁵or about 1×10⁵ plaque forming units (PFU), 5×10⁵ or about 5×10⁵ PFU,1×10⁶ or about 1×10⁶ PFU, 5×10⁶ or about 5×10⁶ PFU, 1×10⁷ or about 1×107PFU, 5×10⁷ or about 5×10⁷ PFU, 1×10⁸ or about 1×10⁸ PFU, 5×10⁸ or about5×10⁸ PFU, 1×10⁹ or about 1×10⁹ PFU, 5×10⁹ or about 5×10⁹ PFU, 1×10¹⁰ orabout 1×10¹⁰ PFU or 5×10¹⁰ or about 5×10¹⁰ PFU as total single dosagefor an average human of 75 kg or adjusted for the weight and size andspecies of the subject. One of skill in the art can determine suitabledosage. Suitable dosages for a virus can be determined empirically.

Also useful in the practice of the disclosure is an adenovirus vector.One advantage is the ability of recombinant adenoviruses to efficientlytransfer and express recombinant genes in a variety of mammalian cellsand tissues in vitro and in vivo, resulting in the high expression ofthe transferred nucleic acids. Further, the ability to productivelyinfect quiescent cells, expands the utility of recombinant adenoviralvectors. In addition, high expression levels ensure that the products ofthe nucleic acids will be expressed to sufficient levels to generate animmune response (see e.g., U.S. Pat. No. 7,029,848, hereby incorporatedby reference). As to adenovirus vectors useful in the practice of thedisclosure, mention is made of U.S. Pat. No. 6,955,808. The adenovirusvector used can be selected from the group consisting of the Ad5, Ad35,Ad11, C6, and C7 vectors. The sequence of the Adenovirus 5 (“Ad5”)genome has been published. (Chroboczek, J., Bieber, F., and Jacrot, B.(1992) The Sequence of the Genome of Adenovirus Type 5 and ItsComparison with the Genome of Adenovirus Type 2, Virology 186, 280-285;the contents if which is hereby incorporated by reference). Ad35 vectorsare described in U.S. Pat. Nos. 6,974,695, 6,913,922, and 6,869,794.Ad11 vectors are described in U.S. Pat. No. 6,913,922. C6 adenovirusvectors are described in U.S. Pat. Nos. 6,780,407; 6,537,594; 6,309,647;6,265,189; 6,156,567; 6,090,393; 5,942,235 and 5,833,975. C7 vectors aredescribed in U.S. Pat. No. 6,277,558. Adenovirus vectors that areE1-defective or deleted, E3-defective or deleted, and/or E4-defective ordeleted can also be used. Certain adenoviruses having mutations in theE1 region have improved safety margin because E1-defective adenovirusmutants are replication-defective in non-permissive cells, or, at thevery least, are highly attenuated. Adenoviruses having mutations in theE3 region can have enhanced the immunogenicity by disrupting themechanism whereby adenovirus down-regulates MHC class I molecules.Adenoviruses having E4 mutations can have reduced immunogenicity of theadenovirus vector because of suppression of late gene expression. Suchvectors can be particularly useful when repeated re-vaccinationutilizing the same vector is desired. Adenovirus vectors that aredeleted or mutated in E1, E3, E4, E1 and E3, and E1 and E4 can be usedin accordance with the present disclosure. Furthermore, “gutless”adenovirus vectors, in which all viral genes are deleted, can also beused in accordance with the present disclosure. Such vectors require ahelper virus for their replication and require a special human 293 cellline expressing both E1a and Cre, a condition that does not exist innatural environment. Such “gutless” vectors are non-immunogenic and thusthe vectors can be inoculated multiple times for re-vaccination. The“gutless” adenovirus vectors can be used for insertion of heterologousinserts/genes such as the transgenes of the present disclosure, and caneven be used for co-delivery of a large number of heterologousinserts/genes. In some embodiments, the delivery is via an adenovirus,which can be at a single booster dose. In some embodiments, theadenovirus is delivered via multiple doses. In terms of in vivodelivery, AAV is advantageous over other viral vectors due to lowtoxicity and low probability of causing insertional mutagenesis becauseit doesn't integrate into the host genome. AAV has a packaging limit of4.5 or 4.75 Kb. Constructs larger than 4.5 or 4.75 Kb result insignificantly reduced virus production. There are many promoters thatcan be used to drive nucleic acid molecule expression. AAV ITR can serveas a promoter and is advantageous for eliminating the need for anadditional promoter element. For ubiquitous expression, the followingpromoters can be used: CMV, CAG, CBh, PGK, SV40, Ferritin heavy or lightchains, etc. For brain expression, the following promoters can be used:Synapsinl for all neurons, CaMKIIalpha for excitatory neurons, GAD67 orGAD65 or VGAT for GABAergic neurons, etc. Promoters used to drive RNAsynthesis can include: Pol III promoters such as U6 or Hl. The use of aPol II promoter and intronic cassettes can be used to express guide RNA(gRNA). With regard to AAV vectors useful in the practice of thedisclosure, mention is made of U.S. Pat. Nos. 5,658,785, 7,115,391,7,172,893, 6,953,690, 6,936,466, 6,924,128, 6,893,865, 6,793,926,6,537,540, 6,475,769 and 6,258,595, and documents cited therein. As toAAV, the AAV can be AAV1, AAV2, AAV5 or any combination thereof. One canselect the AAV with regard to the cells to be targeted; e.g., one canselect AAV serotypes 1, 2, 5 or a hybrid capsid AAV1, AAV2, AAV5 or anycombination thereof for targeting brain or neuronal cells; and one canselect AAV4 for targeting cardiac tissue. AAV8 is useful for delivery tothe liver. In some embodiments the delivery is via an AAV. The dosagecan be adjusted to balance the therapeutic benefit against any sideeffects.

In some embodiments, effectively activating a cellular immune responsefor a disease vaccine or immunogenic composition can be achieved byexpressing the relevant antigens in a vaccine or immunogenic compositionin a non-pathogenic microorganism. Well-known examples of suchmicroorganisms are Mycobacterium bovis BCG, Salmonella and Pseudomonas(See, U.S. Pat. No. 6,991,797, hereby incorporated by reference in itsentirety).

In some embodiments, a Poxvirus is used in the disease vaccine orimmunogenic composition. These include orthopoxvirus, avipox, vaccinia,MVA, NYVAC, canarypox, ALVAC, fowlpox, TROVAC, etc. (see e.g., Verardiet al., Hum Vaccin Immunother. 2012 July; 8(7):961-70; and Moss,Vaccine. 2013; 31(39): 4220-4222). Poxvirus expression vectors weredescribed in 1982 and quickly became widely used for vaccine developmentas well as research in numerous fields. Advantages of the vectorsinclude simple construction, ability to accommodate large amounts offoreign DNA and high expression levels. Information concerningpoxviruses that can be used in the practice of the disclosure, such asChordopoxvirinae subfamily poxviruses (poxviruses of vertebrates), forinstance, orthopoxviruses and avipoxviruses, e.g., vaccinia virus (e.g.,Wyeth Strain, WR Strain (e.g., ATCC® VR-1354), Copenhagen Strain, NYVAC,NYVAC.1, NYVAC.2, MVA, MVA-BN), canarypox virus (e.g., Wheatley C93Strain, ALVAC), fowlpox virus (e.g., FP9 Strain, Webster Strain,TROVAC), dovepox, pigeonpox, quailpox, and raccoon pox, inter alia,synthetic or non-naturally occurring recombinants thereof, uses thereof,and methods for making and using such recombinants can be found inscientific and patent literature.

In some embodiments, the vaccinia virus is used in the disease vaccineor immunogenic composition to express a antigen. (Rolph et al.,Recombinant viruses as vaccines and immunological tools. Curr OpinImmunol 9:517-524, 1997). The recombinant vaccinia virus is able toreplicate within the cytoplasm of the infected host cell and thepolypeptide of interest can therefore induce an immune response.Moreover, Poxviruses have been widely used as vaccine or immunogeniccomposition vectors because of their ability to target encoded antigensfor processing by the major histocompatibility complex class I pathwayby directly infecting immune cells, in particular antigen-presentingcells, but also due to their ability to self-adjuvant.

In some embodiments, ALVAC is used as a vector in a disease vaccine orimmunogenic composition. ALVAC is a canarypox virus that can be modifiedto express foreign transgenes and has been used as a method forvaccination against both prokaryotic and eukaryotic antigens (Honig H,Lee D S, Conkright W, et al. Phase I clinical trial of a recombinantcanarypoxvirus (ALVAC) vaccine expressing human carcinoembryonic antigenand the B7.1 co-stimulatory molecule. Cancer Immunol Immunother 2000;49:504-14; von Mehren M, Arlen P, Tsang K Y, et al. Pilot study of adual gene recombinant avipox vaccine containing both carcinoembryonicantigen (CEA) and B7.1 transgenes in patients with recurrentCEA-expressing adenocarcinomas. Clin Cancer Res 2000; 6:2219-28; MuseyL, Ding Y, Elizaga M, et al. HIV-1 vaccination administeredintramuscularly can induce both systemic and mucosal T cell immunity inHIV-1-uninfected individuals. J Immunol 2003; 171:1094-101; Paoletti E.Applications of pox virus vectors to vaccination: an update. Proc NatlAcad Sci USA 1996; 93:11349-53; U.S. Pat. No. 7,255,862). In a phase Iclinical trial, an ALVAC virus expressing the tumor antigen CEA showedan excellent safety profile and resulted in increased CEA-specificT-cell responses in selected patients; objective clinical responses,however, were not observed (Marshall J L, Hawkins M J, Tsang K Y, et al.Phase I study in cancer patients of a replication-defective avipoxrecombinant vaccine that expresses human carcinoembryonic antigen. JClin Oncol 1999; 17:332-7).

In some embodiments, a Modified Vaccinia Ankara (MVA) virus can be usedas a viral vector for an antigen vaccine or immunogenic composition. MVAis a member of the Orthopoxvirus family and has been generated by about570 serial passages on chicken embryo fibroblasts of the Ankara strainof Vaccinia virus (CVA) (for review see Mayr, A., et al., Infection 3,6-14, 1975). As a consequence of these passages, the resulting MVA viruscontains 31 kilobases less genomic information compared to CVA, and ishighly host-cell restricted (Meyer, H. et al., J. Gen. Virol. 72,1031-1038, 1991). MVA is characterized by its extreme attenuation,namely, by a diminished virulence or infectious ability, but still holdsan excellent immunogenicity. When tested in a variety of animal models,MVA was proven to be avirulent, even in immuno-suppressed individuals.Moreover, MVA-BN®-HER2 is a candidate immunotherapy designed for thetreatment of HER-2-positive breast cancer and is currently in clinicaltrials. (Mandl et al., Cancer Immunol Immunother. January 2012; 61(1):19-29). Methods to make and use recombinant MVA has been described(e.g., see U.S. Pat. Nos. 8,309,098 and 5,185,146 hereby incorporated inits entirety).

In some embodiments, recombinant viral particles of the vaccine orimmunogenic composition are administered to patients in need thereof.

Provided herein is a method of developing an therapeutic for a subjectwith a disease or condition comprising providing a population of cellsderived from a subject with a disease or condition, expressing in one ormore cells of the population of cells an affinity acceptor tagged classI or class II HLA allele by introducing into the one or more cells apolynucleic acid encoding a sequence comprising: a sequence encoding arecombinant class I or class II HLA allele operatively linked to asequence encoding an affinity acceptor peptide, thereby forming affinityacceptor tagged HLA-peptide complexes in the one or more cells;enriching and characterizing the affinity acceptor tagged HLA-peptidecomplexes; and, optionally developing an therapeutic based on thecharacterization.

Provided herein is a method of identifying at least one subject specificimmunogenic antigen and preparing a subject-specific immunogeniccomposition that includes the at least one subject specific immunogenicantigen, wherein the subject has a disease and the at least one subjectspecific immunogenic antigen is specific to the subject and thesubject's disease, said method comprising: providing a population ofcells derived from a subject with a disease or condition, expressing inone or more cells of the population of cells from the subject, anaffinity acceptor tagged class I or class II HLA allele by introducinginto the one or more cells a polynucleic acid encoding a sequencecomprising: a sequence encoding a recombinant class I or class II HLAallele operatively linked to a sequence encoding an affinity acceptorpeptide, thereby forming affinity acceptor tagged HLA-peptide complexesin the one or more cells; enriching affinity acceptor tagged HLA-peptidecomplexes from the one or more cells; identifying an immunogenic peptidefrom the enriched affinity acceptor tagged HLA-peptide complexes that isspecific to the subject and the subject's disease; and formulating asubject-specific immunogenic composition based one or more of thesubject specific immunogenic peptides identified.

In some embodiments, the therapeutic or subject specific immunogeniccomposition comprises a peptide from the enriched affinity acceptortagged HLA-peptide complexes or a or a polynucleotide encoding thepolypeptide from the enriched affinity acceptor tagged HLA-peptidecomplexes.

In some embodiments, the therapeutic or subject specific immunogeniccomposition comprises a T cell expressing a T cell receptor (TCR) thatspecifically binds to the polypeptide from the enriched affinityacceptor tagged HLA-peptide complexes. In some embodiments, the subjectspecific immunogenic composition comprises a chimeric antigen receptor(CAR) T cell expressing a receptor that specifically binds to thepolypeptide from the enriched affinity acceptor tagged HLA-peptidecomplexes. In some embodiments, the method further comprisesadministering another therapeutic agent, optionally, an immunecheckpoint inhibitor to the subject. In some embodiments, the methodfurther comprises administering an adjuvant, optionally, poly-ICLC tothe subject.

In some embodiments, the disease or disorder is cancer. In someembodiments, the disease or disorder is an autoimmune disease. In someembodiments, the disease or disorder is an infection. In someembodiments, the infection is an infection by an infectious agent. Insome embodiments, the infectious agent is a pathogen, a virus, bacteria,or a parasite. In some embodiments, the virus is selected from the groupconsisting of: BK virus (BKV), Dengue viruses (DENV-1, DENV-2, DENV-3,DENV-4, DENV-5), cytomegalovirus (CMV), Hepatitis B virus (HBV),Hepatitis C virus (HCV), Epstein-Barr virus (EBV), an adenovirus, humanimmunodeficiency virus (HIV), human T-cell lymphotrophic virus (HTLV-1),an influenza virus, RSV, HPV, rabies, mumps rubella virus, poliovirus,yellow fever, hepatitis A, hepatitis B, Rotavirus, varicella virus,human papillomavirus (HPV), smallpox, zoster, and any combinationthereof. In some embodiments, the bacteria is selected from the groupconsisting of: Klebsiella spp., Tropheryma whipplei, Mycobacteriumleprae, Mycobacterium lepromatosis, and Mycobacterium tuberculosis,typhoid, pneumococcal, meningococcal, haemophilus B, anthrax, tetanustoxoid, meningococcal group B, bcg, cholera, and combinations thereof.In some embodiments, the parasite is a helminth or a protozoan. In someembodiments, the parasite is selected from the group consisting of:Leishmania spp., Plasmodium spp., Trypanosoma cruzi, Ascarislumbricoides, Trichuris trichiura, Necator americanus, Schistosoma spp.,and any combination thereof.

Provided herein is a method of developing a therapeutic for a subjectwith a disease or condition comprising: providing a population of cells,wherein one or more cells of the population of cells comprise apolynucleic acid comprising a sequence encoding at least two affinityacceptor tagged class I or class II HLA alleles, wherein the sequenceencoding the at least two affinity acceptor tagged class I or class IIHLAs comprises a first recombinant sequence comprising a sequenceencoding a first class I or class II HLA allele operatively linked to asequence encoding a first affinity acceptor peptide; and a secondrecombinant sequence comprising a sequence encoding a second class I orclass II HLA allele operatively linked to a sequence encoding a secondaffinity acceptor peptide; expressing the at least two affinity acceptortagged HLAs in at least one cell of the one or more cells of thepopulation of cells, thereby forming affinity acceptor taggedHLA-peptide complexes in the at least one cell; enriching for theaffinity acceptor tagged HLA-peptide complexes; and identifying apeptide from the enriched affinity acceptor tagged HLA-peptidecomplexes; and formulating an immunogenic composition based one or moreof the peptides identified, wherein the first and the second recombinantclass I or class II HLA alleles are matched to an HLA haplotype of asubject.

In some embodiments, the subject has a disease or condition. In someembodiments, the first recombinant class I or class II HLA allele isdifferent than the second recombinant class I or class II HLA allele. Insome embodiments, the first affinity acceptor peptide is the same as thesecond affinity acceptor peptide. In some embodiments, the methodcomprises characterizing a peptide bound to the first and/or secondaffinity acceptor tagged HLA-peptide complexes from the enriching. Insome embodiments, the at least two affinity acceptor tagged class I orclass II HLA alleles comprise at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50 class I and/orclass II HLA alleles. In some embodiments, the first and/or the secondaffinity acceptor tagged HLA-peptide complexes comprise a transmembranedomain. In some embodiments, the first and/or the second affinityacceptor tagged HLA-peptide complexes comprise an intracellular domain.In some embodiments, the first and/or the second affinity acceptortagged HLA-peptide complexes are not excreted. In some embodiments, thefirst and/or the second affinity acceptor tagged HLA-peptide complexesincorporate into a cell membrane when expressed. In some embodiments,the first and/or the second affinity acceptor tagged HLA-peptidecomplexes are not soluble affinity acceptor tagged HLA-peptidecomplexes. In some embodiments, the method further comprises generatingan HLA-allele specific peptide database. In some embodiments, the methodcomprises introducing one or more exogenous peptides to the populationof cells. In some embodiments, the introducing comprises contacting thepopulation of cells with the one or more exogenous peptides orexpressing the one or more exogenous peptides in the population ofcells. In some embodiments, the introducing comprises contacting thepopulation of cells with one or more nucleic acids encoding the one ormore exogenous peptides. In some embodiments, the one or more nucleicacids encoding the one or more peptides is DNA. In some embodiments, theone or more nucleic acids encoding the one or more peptides is RNA,optionally wherein the RNA is mRNA. In some embodiments, the enrichingdoes not comprise use of a tetramer reagent. In some embodiments, themethod comprises determining the sequence of a peptide or a portionthereof bound to the first and/or the second affinity acceptor taggedHLA-peptide complex from the enriching. In some embodiments, thedetermining comprises biochemical analysis, mass spectrometry analysis,MS analysis, MS/MS analysis, LC-MS/MS analysis, or a combinationthereof. In some embodiments, the method comprises evaluating a bindingaffinity or stability of a peptide or a portion thereof bound to thefirst and/or the second affinity acceptor tagged HLA-peptide complexfrom the enriching.

In some embodiments, the method comprises determining whether a peptideor a portion thereof bound to the first and/or the second affinityacceptor tagged HLA-peptide complex from the enriching contains one ormore mutations. In some embodiments, the method comprises evaluatingassociations of peptides with HLA molecules in the first and/or thesecond affinity acceptor tagged HLA-peptide complex.

In some embodiments, the method comprises expressing a library ofpeptides in the population of cells, thereby forming a library ofaffinity acceptor tagged HLA-peptide complexes. In some embodiments, themethod comprises contacting to the population of cells a library ofpeptides or a library of sequences encoding peptides, thereby forming alibrary of affinity acceptor tagged HLA-peptide complexes. In someembodiments, the library comprises a library of peptides associated witha disease or condition. In some embodiments, the disease or condition iscancer or an infection with an infectious agent.

In some embodiments, the method comprises introducing the infectiousagent or portions thereof into one or more cells of the population ofcells. In some embodiments, the method comprises characterizing one ormore peptides from the first and/or the second HLA-peptide complexes,optionally wherein the peptides are from one or more target proteins ofthe infectious agent. In some embodiments, the method comprisescharacterizing one or more regions of the peptides from the one or moretarget proteins of the infectious agent. In some embodiments, the methodcomprises identifying peptides from the first and/or the secondHLA-peptide complexes derived from an infectious agent.

In some embodiments, the population of cells is from a biological samplefrom a subject with a disease or condition. In some embodiments, thepopulation of cells is a cell line. In some embodiments, the populationof cells is a population of primary cells.

In some embodiments, the peptide from the first and/or the secondaffinity acceptor tagged HLA-peptide complex is capable of activating aT cell from a subject when presented by an antigen presenting cell. Insome embodiments, the method comprises comparing HLA-peptide complexesfrom diseased cells to HLA-peptide complexes from non-diseased cells.

In some embodiments, the method further comprises isolating peptidesfrom the first and/or the second affinity acceptor tagged HLA-peptidecomplexes before the identifying.

In some embodiments, the population of cells is a population of low cellsurface HLA class I or class II expressing cells. In some embodiments,the population of cells expresses one or more endogenous HLA alleles. Insome embodiments, the population of cells is an engineered population ofcells lacking one or more endogenous HLA class I alleles. In someembodiments, the population of cells is an engineered population ofcells lacking endogenous HLA class I alleles. In some embodiments, thepopulation of cells is an engineered population of cells lacking one ormore endogenous HLA class II alleles. In some embodiments, thepopulation of cells is an engineered population of cells lackingendogenous HLA class II alleles. In some embodiments, the population ofcells is an engineered population of cells lacking endogenous HLA classI alleles and endogenous HLA class II alleles. In some embodiments, thepopulation of cells is a knock-out of one or more HLA class I alleles.In some embodiments, the population of cells is a knock-out of one ormore HLA class II alleles. In some embodiments, the population of cellsis a knock-out of all HLA class I alleles.

In some embodiments, the population of cells is a knock-out of all HLAclass II alleles. In some embodiments, the population of cells is aknock-out of all HLA class I alleles and a knock-out of all HLA class IIalleles.

In some embodiments, the sequence encoding the at least two affinityacceptor tagged class I or class II HLA alleles encodes a class I HLA.In some embodiments, the class I HLA is selected from the groupconsisting of HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, and HLA-G. In someembodiments, the first recombinant class I or class II HLA allele is afirst class I HLA allele and the second recombinant class I or class IIHLA allele is a second class I HLA allele. In some embodiments, thesequence encoding the at least two affinity acceptor tagged class I orclass II HLA alleles encodes a class II HLA. In some embodiments, theclass II HLA is selected from the group consisting of HLA-DR, HLA-DQ,and HLA-DP. In some embodiments, the class II HLA comprises a HLA classII α-chain, a HLA class II β-chain, or a combination thereof. In someembodiments, the first recombinant class I or class II HLA allele is afirst class II HLA allele and the second recombinant class I or class IIHLA allele is a second class II HLA allele.

In some embodiments, the first sequence and the second sequence are eachoperatively linked. In some embodiments, the first sequence and thesecond sequence are comprised on different polynucleotide molecules.

In some embodiments, the sequence encoding the first and/or secondaffinity acceptor peptide is operatively linked to a sequence thatencodes an extracellular portion of the first and/or second class I orclass II HLA allele. In some embodiments, the first and/or secondencoded affinity acceptor peptide is expressed extracellularly. In someembodiments, the sequence encoding the first and/or second affinityacceptor peptide is operatively linked to the N-terminus of the sequenceencoding the first and/or second class I or class II HLA allele.

In some embodiments, the sequence encoding the first and/or secondaffinity acceptor peptide is operatively linked to a sequence thatencodes an intracellular portion of the first and/or second class I orclass II HLA allele. In some embodiments, the encoded first and/orsecond affinity acceptor peptide is expressed intracellularly. In someembodiments, the sequence encoding the first and/or second affinityacceptor peptide is operatively linked to the C-terminus of the sequenceencoding the first and/or second class I or class II HLA allele. In someembodiments, the sequence encoding the first and/or second affinityacceptor peptide is operatively linked to the sequence encoding thefirst and/or second class I or class II HLA allele by a linker.

In some embodiments, enriching comprises enriching for intact cellsexpressing the first and/or second affinity acceptor tagged HLA-peptidecomplexes. In some embodiments, the method does not comprise lysing thecells before enriching. In some embodiments, the method furthercomprises lysing the one or more cells before enriching.

In some embodiments, enriching comprises contacting an affinity acceptorpeptide binding molecule to the first and/or second affinity acceptortagged HLA-peptide complexes, wherein the affinity acceptor peptidebinding molecule binds specifically to the first and/or second affinityacceptor peptide. In some embodiments, the first and/or second affinityacceptor peptide comprises a tag sequence comprising a biotin acceptorpeptide (BAP), poly-histidine tag, poly-histidine-glycine tag,poly-arginine tag, poly-aspartate tag, poly-cysteine tag,poly-phenylalanine, c-myc tag, Herpes simplex virus glycoprotein D (gD)tag, FLAG tag, KT3 epitope tag, tubulin epitope tag, T7 gene 10 proteinpeptide tag, streptavidin tag, streptavidin binding peptide (SPB) tag,Strep-tag, Strep-tag II, albumin-binding protein (ABP) tag, alkalinephosphatase (AP) tag, bluetongue virus tag (B-tag), calmodulin bindingpeptide (CBP) tag, chloramphenicol acetyl transferase (CAT) tag,choline-binding domain (CBD) tag, chitin binding domain (CBD) tag,cellulose binding domain (CBP) tag, dihydrofolate reductase (DHFR) tag,galactose-binding protein (GBP) tag, maltose binding protein (MBP),glutathione-S-transferase (GST), Glu-Glu (EE) tag, human influenzahemagglutinin (HA) tag, horseradish peroxidase (HRP) tag, NE-tag, HSVtag, ketosteroid isomerase (KSI) tag, KT3 tag, LacZ tag, luciferase tag,NusA tag, PDZ domain tag, AviTag, Calmodulin-tag, E-tag, S-tag, SBP-tag,Softag 1, Softag 3, TC tag, VSV-tag, Xpress tag, Isopeptag, SpyTag,SnoopTag, Profinity eXact tag, Protein C tag, S1-tag, S-tag,biotin-carboxy carrier protein (BCCP) tag, green fluorescent protein(GFP) tag, small ubiquitin-like modifier (SUMO) tag, tandem affinitypurification (TAP) tag, HaloTag, Nus-tag, Thioredoxin-tag, Fc-tag, CYDtag, HPC tag, TrpE tag, ubiquitin tag, VSV-G epitope tag, V5 tag, or acombination thereof optionally, wherein the first and/or second affinityacceptor peptide comprises two or more repeats of a tag sequence. Insome embodiments, the affinity acceptor peptide binding molecule isbiotin or an antibody specific to the first and/or second affinityacceptor peptide.

In some embodiments, the enriching comprises contacting an affinitymolecule to the first and/or second affinity acceptor tagged HLA-peptidecomplexes, wherein the affinity molecule binds specifically to theaffinity acceptor peptide binding molecule. In some embodiments, theaffinity molecule is streptavidin, NeutrAvidin, or a derivative thereof.In some embodiments, enriching comprises immunoprecipitating the firstand/or second affinity acceptor tagged HLA-peptide complexes. In someembodiments, the affinity acceptor peptide binding molecule is attachedto a solid surface. In some embodiments, the affinity molecule isattached to a solid surface. In some embodiments, the solid surface is abead.

In some embodiments, enriching comprises immunoprecipitating the firstand/or second affinity acceptor tagged HLA-peptide complexes with anaffinity acceptor peptide binding molecule that binds specifically tothe first and/or second affinity acceptor peptide. In some embodiments,the affinity acceptor peptide binding molecule does not specificallyinteract with the amino acid sequence of the encoded first and/or secondclass I or class II HLA. In some embodiments, enriching comprisescontacting an affinity molecule specific to an extracellular portion ofthe first and/or second class I or class II HLA allele. In someembodiments, enriching comprises contacting an affinity moleculespecific to an N-terminal portion of the first and/or second class I orclass II HLA allele.

In some embodiments, providing comprises contacting the population ofcells with the polynucleic acid. In some embodiments, contactingcomprises transfecting or transducing. In some embodiments, providingcomprises contacting the population of cells with a vector comprisingthe polynucleic acid. In some embodiments, the vector is a viral vector.In some embodiments, the polynucleic acid is stably integrated into thegenome of the population of cells.

In some embodiments, the sequence encoding the first and/or second classI or class II HLA comprises a sequence encoding a HLA class I α-chain.In some embodiments, the first recombinant class I or class II HLAallele is a first HLA class I α-chain and the second recombinant class Ior class II HLA allele is a second HLA class I α-chain. In someembodiments, the method further comprises expressing a sequence encodingβ2 microglobulin in the one or more cells. In some embodiments, thesequence encoding β2 microglobulin is connected to the sequence encodingthe first and/or second class I or class II HLA. In some embodiments,the sequence encoding β2 microglobulin is connected to the sequenceencoding the first and/or second class I or class II HLA by a linker. Insome embodiments, the sequence encoding β2 microglobulin is connected toa sequence encoding a third affinity acceptor peptide. In someembodiments, the third affinity acceptor peptide is different than thefirst and/or second affinity acceptor peptide.

In some embodiments, the sequence encoding the first and/or second classI or class II HLA comprises a sequence encoding a HLA class II α-chainand/or a HLA class II β-chain. In some embodiments, the sequenceencoding the first and/or second class I or class II HLA comprises asequence encoding a first HLA class II α-chain and a second HLA class IIα-chain. In some embodiments, the method further comprises expressing asequence encoding a HLA class II β-chain in the one or more cells. Insome embodiments, the sequence encoding a first HLA class II α-chain anda second HLA class II α-chain HLA is connected to the sequence encodingthe HLA class II β-chain. In some embodiments, the sequence encoding thefirst and/or second class I or class II HLA comprises a sequenceencoding a first HLA class II β-chain and a second HLA class II β-chain.In some embodiments, the method further comprises expressing a sequenceencoding a HLA class II α-chain in the one or more cells. In someembodiments, the sequence encoding a first HLA class II β-chain and asecond HLA class II β-chain is connected to the sequence encoding theHLA class II α-chain by a linker. In some embodiments, the sequenceencoding the HLA class II β-chain or the HLA class II α-chain isconnected to a sequence encoding a third affinity acceptor peptide. Insome embodiments, the third affinity acceptor peptide is different thanthe first and/or second affinity acceptor peptide.

In some embodiments, the third affinity acceptor peptide is differentthan the first affinity acceptor peptide and is selected from the groupconsisting of biotin acceptor peptide (BAP), poly-histidine tag,poly-histidine-glycine tag, poly-arginine tag, poly-aspartate tag,poly-cysteine tag, poly-phenylalanine, c-myc tag, Herpes simplex virusglycoprotein D (gD) tag, FLAG tag, KT3 epitope tag, tubulin epitope tag,T7 gene 10 protein peptide tag, streptavidin tag, streptavidin bindingpeptide (SPB) tag, Strep-tag, Strep-tag II, albumin-binding protein(ABP) tag, alkaline phosphatase (AP) tag, bluetongue virus tag (B-tag),calmodulin binding peptide (CBP) tag, chloramphenicol acetyl transferase(CAT) tag, choline-binding domain (CBD) tag, chitin binding domain (CBD)tag, cellulose binding domain (CBP) tag, dihydrofolate reductase (DHFR)tag, galactose-binding protein (GBP) tag, maltose binding protein (MBP),glutathione-S-transferase (GST), Glu-Glu (EE) tag, human influenzahemagglutinin (HA) tag, horseradish peroxidase (HRP) tag, NE-tag, HSVtag, ketosteroid isomerase (KSI) tag, KT3 tag, LacZ tag, luciferase tag,NusA tag, PDZ domain tag, AviTag, Calmodulin-tag, E-tag, S-tag, SBP-tag,Softag 1, Softag 3, TC tag, VSV-tag, Xpress tag, Isopeptag, SpyTag,SnoopTag, Profinity eXact tag, Protein C tag, S1-tag, S-tag,biotin-carboxy carrier protein (BCCP) tag, green fluorescent protein(GFP) tag, small ubiquitin-like modifier (SUMO) tag, tandem affinitypurification (TAP) tag, HaloTag, Nus-tag, Thioredoxin-tag, Fc-tag, CYDtag, HPC tag, TrpE tag, ubiquitin tag, VSV-G epitope tag, V5 tag, and acombination thereof; optionally, wherein the first or second affinityacceptor peptide comprises two or more repeats of a tag sequence.

In some embodiments, the linker comprises a polynucleic acid sequenceencoding a cleavable linker. In some embodiments, the cleavable linkeris a ribosomal skipping site or an internal ribosomal entry site (IRES)element. In some embodiments, the ribosomal skipping site or IRES iscleaved when expressed in the cells. In some embodiments, the ribosomalskipping site is selected from the group consisting of F2A, T2A, P2A,and E2A. In some embodiments, the IRES element is selected from commoncellular or viral IRES sequences.

In some embodiments, the method comprises performing biochemicalanalysis or mass spectrometry, such as tandem mass spectrometry.

In some embodiments, the method comprises obtaining a peptide sequencethat corresponds to an MS/MS spectra of one or more peptides isolatedfrom the enriched affinity acceptor tagged HLA-peptide complexes from apeptide database; wherein one or more sequences obtained identifies thesequence of the one or more peptides.

In some embodiments, the population of cells is a cell line selectedfrom HEK293T, expi293, HeLa, A375, 721.221, JEG-3, K562, Jurkat, Hep G2,SH-SY5Y, CACO-2, U937, U-2 OS, ExpiCHO, CHO and THP1.

In some embodiments, the cell line is treated with one or morecytokines, checkpoint inhibitors, epigenetically-active drugs, IFN-γ, ora combination thereof.

In some embodiments, the population of cells comprises at least 10⁵cells, at least 10⁶ cells or at least 10⁷ cells. In some embodiments,the population of cells is a population of dendritic cells, macrophages,cancer cells or B-cells. In some embodiments, the population of cellscomprises tumor cells.

In some embodiments, the population of cells is contacted with an agentprior to isolating the first and/or second HLA-peptide complexes fromthe one or more cells. In some embodiments, the agent is an inflammatorycytokine, a chemical agent, an adjuvant, a therapeutic agent orradiation.

In some embodiments, the first and or second HLA allele is a mutated HLAallele. In some embodiments, the sequence encoding the first and orsecond HLA allele comprises a barcode sequence. In some embodiments, themethod further comprises assaying for expression of the first and/orsecond affinity acceptor tagged class I or class II HLA allele. In someembodiments, the assaying comprises sequencing the first and/or secondaffinity acceptor tagged class I or class II HLA allele, detecting RNAencoding the first and/or second affinity acceptor tagged class I orclass II HLA allele RNA, detecting the first and/or second affinityacceptor tagged class I or class II HLA allele protein, or a combinationthereof. In some embodiments, the first and second affinity acceptortagged class I or class II HLA allele comprises a unique barcodesequence. In some embodiments, the first sequence and the secondsequence comprise a unique barcode sequence.

EXAMPLES

The examples provided below are for illustrative purposes only and donot to limit the scope of the claims provided herein.

Example 1. Universal IP Pipeline: Universal Single-Allelic HLA-PeptideComplex Identification Platform

Universal immunopurification (IP) constructs disclosed herein consist ofa DNA construct coding for affinity-tagged HLA class I or class IIalleles that are expressed off a mammalian expression vector viacellular transfection or transduction (FIG. 1A and FIG. 1B).Non-limiting exemplary class I and class II HLA constructs are shown inFIG. 2. Non-limiting exemplary affinity tags include the biotin acceptorpeptide (BAP) or Human influenza hemagglutinin (HA) peptide sequence.The affinity tags can be placed on either the N-terminus or C-terminusof the HLA allele. A cleavage sequence, such as F2A shown in FIG. 2, oran internal ribosome entry site (IRES) can be placed between the α-chainand β2-microglobulin (class I) or between the α-chain and β-chain (classII). Non-limiting exemplary vectors include a lentiviral vector as shownin FIG. 3. Antibody resistance genes, such as puromycin resistance(Puro), are incorporated into the constructs to allow for selectionafter transfection or transduction. Cells transfected or transduced withUniversal IP constructs are either expanded (FIG. 4A) or selected andthen expanded (FIG. 4B) prior to LC-MS/MS analyses. Schematics ofuniversal immunopurification platform for class I and class II HLA areshown in FIG. 5.

Example 2. Cell Culture and HLA-Peptide Immunopurification andSequencing

Mono-allelic HLA cells were generated by transducing B721.221, A375,JEG-3, K562, Jurkat, or HEK293T, HeLa, or expi293 cells with aretroviral vector coding a single class I HLA allele (e.g., HLA-A*02:01,HLA-A*23:01 and HLA-B*14:02, or HLA-E*01:01) or class II HLA allele(e.g., HLA-DRB*01:01, HLA-DRB*01:02 and HLA-DRB*11:01, or HLA-DRB*15:01,or HLA-DRB*07:01) as described previously (Reche et al., 2006). Theclass I or II HLA-types of cell lines were confirmed by standardmolecular typing. The cells were cultured and HLA-peptideimmunopurification was performed.

Proof of concept transduction of class I HLA alleles (FIG. 6C) intoHEK293T cells is shown in FIGS. 6A-6C. Mock, GFP, and empty plasmidtransductions with HLA-A*02:01 constructs for biotinylation-baseduniversal immunopurification was performed and biotinylation wasconfirmed in a Western blot (FIG. 6A). A Ponceau stained gel was used asa loading control for the Western blot analysis (FIG. 6B). Transfectionand biotinylation optimization of class I and class II HLA-BAP alleles(FIG. 7C) expressed by HEK293T cells are shown in FIGS. 7A-7C. Abiotinylation time course experiment showed that C- and N-terminallylabeled HLA-BAP biotinylation was complete in 10 minutes for both classI and class II HLA-BAP expressing cells (FIG. 7A and FIG. 7B)

The presently disclosed universal IP pipeline was tested in multiplecell types (FIGS. 8A-8D). The universal IP constructs for both class Iand class II HLA were transfected into HEK293T (human embryonic kidney)(FIG. 8A), HeLa (human cervical cancer) (FIG. 8B), A375 (human malignantmelanoma) (FIG. 8C), and Expi293 (human embryonic kidney geneticallyengineered for high density culture and protein expression) cells (FIG.8D). Western blots were performed using anti-streptavidin for BAP labeland anti-HA for HA label, and a Ponceau stained gel was used as aloading control for the Western blots. The Western blots confirmed theexpressions of both class I and class II constructs in all cell typestested (FIGS. 8A-8D).

The following describes materials and methods used in this Example.

Universal IP of Class I and Class H HLA Alleles (Biotin)

Cells were transfected or transduced to express Universal IP constructsfollowing standard methods. After transduction, the cells areresuspended in the media and transferred to a 50 ml falcon tubes. Thetubes were spun at 1500 rpm for 5 minutes and the media was removed. Thecells were then resuspended in 1.5 ml of cold PBS and transferred to a1.5 mL Eppendorf tube. The tubes were then centrifuged (550×g at 4° C.)for 5 minutes. The PBS was removed and the cells were then resuspendedin 1.2 ml lysis buffer. The cells were resuspended in the bufferfollowed by the addition of benzonase. The tubes were incubated on icewith occasional mixing. After 15 minutes incubating on ice, the tubeswere centrifuged (15,000×g at 4° C.) for 20 minutes. The supernatants(500 μL) were transferred to another 1.5 mL tube (pre-washed) forbiotinylation. Biotinylation of cellular lysates was achieved byaddition of biotin, ATP, and BirA to each sample. The sample was thenincubated at room temperature for 10 minutes and then placed on iceprior to immunoprecipitation.

Immunoprecipitation with NeutrAvidin or streptavidin beads was conductedby addition of pre-washed streptavidin or NeutrAvidin agarose resinslurry to the biotinylated lysate. The sample is then placed on a tuberotisserie, and incubated for 30 minutes at 4° C. After the 30-minuteincubation, the beads are pelleted by centrifugation (1500×g, 1 min, 4°C.) and the supernatant is removed and discarded. The beads were thenresuspended in 1 ml of Wash buffer. The beads were then pelleted bycentrifugation (1,500×g, 1 min, 4° C.) and the wash buffer was removedand discarded. This step was repeated to give a total of four washes inwash buffer. The pelleted beads were resuspended in 1 ml of Tris buffer,pelleted by centrifugation (1,500×g, 1 min, 4° C.), and the Tris bufferwas removed. This step was repeated to give a total of four washes inTris buffer. A final wash was performed in MS grade water byresuspending beads in 1 ml of Mass Spec grade water and centrifuging(1,500×g, 1 min, 4° C.) to pellet the beads. The supernatant was removedand the beads were either stored at −80° C. or immediately subjected toHLA-peptide elution and desalting.

Serial Universal IP of Class H HLA Alleles (HA and Biotin Tagging)

Cells were transfected or transduced to express Universal IP constructsfollowing standard protocols. After transduction, the cells areresuspended in the media and transferred to a 50 ml falcon tubes. Thetubes were spun at 1500 rpm for 5 min and the media was removed. Thecells were then resuspended in 1.5 ml of cold PBS and transferred to a1.5 ml Eppendorf tube. The tubes were then centrifuged (550×g at 4° C.)for 5 minutes. The PBS was removed and the cells were then resuspendedin 1.2 ml lysis buffer. The cells were resuspended in the bufferfollowed by the addition of benzonase. The tubes were incubated on icewith occasional mixing. After 15 minutes incubating on ice, the tubeswere centrifuged (15,000×g at 4° C.) for 20 minutes. The supernatantswere transferred to another 1.5 ml tube (pre-washed) for biotinylation.Biotinylation of cellular lysates was achieved by addition of biotin,ATP, and BirA to each sample. The sample was then incubated at roomtemperature for 10 minutes and then placed on ice prior toimmunoprecipitation.

Immunoprecipitation of HA-tagged class II alleles was carried out byaddition of pre-washed protein G agarose resin that was pre-bound withanti-HA antibody. The sample was then incubated for 60 minutes at 4° C.on a tube rotisserie. After the 60 min incubation, the beads arepelleted by centrifugation (1500×g, 1 min, 4° C.) and the supernatantwas removed and discarded. The beads were washed two times with lysisbuffer and resuspended in lysis buffer containing free HA peptide andincubated for 15 minutes at 4° C. on a tube rotisserie. The beads werethen pelleted by centrifugation (1500×g, 1 min, 4° C.) and thesupernatant was transferred to a 1.5 ml Eppendorf containing 200 ul ofpre-washed NeutrAvidin or streptavidin agarose beads. The sample wasthen placed on a tube rotisserie, and incubated for 30 minutes at 4° C.After the 30 min incubation, the beads are pelleted by centrifugation(1500×g, 1 min, 4° C.) and the supernatant was removed and discarded.The beads were then resuspended in 1 ml of Wash buffer. The beads werethen pelleted by centrifugation (1,500×g, 1 min, 4° C.) and the washbuffer was removed and discarded. This step was repeated to give a totalof four washes in wash buffer. The pelleted beads were resuspended in 1ml of Tris buffer, pelleted by centrifugation (1,500×g, 1 min, 4° C.),and the wash buffer was removed. This step was repeated to give a totalof four washes in the Tris buffer. A final wash was performed in MSgrade water by resuspending beads in 1 ml of Mass Spec grade water andcentrifuging (1,500×g, 1 min, 4° C.) to pellet the beads. Thesupernatant was removed and the beads were either stored at −80° C. orimmediately subjected to HLA-peptide elution and desalting.

HLA-Peptide Elution and Desalting

Peptides were eluted from HLA complexes and desalted on in-house builtEmpore C18 StageTips (3M, 2315) (Rappsilber et al., 2007). Sampleloading, washes, and elution were performed on a tabletop centrifuge ata maximum speed of 1,500-3,000×g. StageTips were equilibrated with twowashes of methanol, two washes of acetonitrile/formic acid, and twowashes of formic acid. In a tube, the dried beads from HLA-associatedpeptide IPs were thawed at 4° C., reconstituted in ACN/formic acidmixture, and loaded onto StageTips. The beads were washed with formicacid, and peptides were further eluted using two rounds of 5 minuteincubations in 10% acetic acid. The combined wash and elution volumeswere combined and loaded onto StageTips. The tubes containing the IPbeads were washed again with formic acid, and this volume was alsoloaded onto StageTips. Peptides were washed twice on StageTips ordesalting cartridges with formic acid. Peptides were eluted using a stepgradient of ACN and formic acid mixtures. Step elutions were combinedand dried to completion.

Example 3. Class I and Class II HLA-Associated Peptide Sequencing byLC-MS/MS

All nano LC-ESI-MS/MS analyses employed the same LC separationconditions described below. Samples were chromatographically separatedusing a Proxeon Easy Nano LC 1000 (Thermo Scientific, San Jose, Calif.)fitted with a PicoFrit 75 μm inner diameter capillary with a 10 μmemitter was packed under pressure to ˜20 cm with of C18 Reprosil beads(1.9 μm particle size, 200 Å pore size, Dr. Maisch GmBH) and heated at50° C. during separation.

Samples were loaded in CAN and formic acid mixture and peptides wereeluted with a linear gradient from 7-30% of Buffer B (either 0.1% FA or0.5% AcOH and 80% or 90% ACN) over 82 min, 30-90% Buffer B over 6 minand then held at 90% Buffer B for 15 min at 200 nL/min (Buffer A, 0.1%FA and 3% ACN) to yield ˜13 (FA) sec peak widths. During data-dependentacquisition, eluted peptides were introduced into either an OrbitrapFusion Lumos Tribrid mass spectrometer (Thermo Scientific) equipped witha nanoelectrospray source at 2.2 kV. A full-scan MS was acquired at aresolution of 30,000 from 300 to 1,800 m/z. Each full scan was followedby top 10 data-dependent MS2 scans at resolution 15,000, using anisolation width of 0.7 m/z.

The numbers of total unique HLA-associated peptides identified frommultiple cell types expressing affinity tagged class I and class II HLAconstructs used in the universal IP pipeline are shown in FIG. 9A. Thenumber of unique peptides from class I HLA mono-allelic peptideprofiling is shown in FIG. 9B. The number of unique peptides from classII HLA mono-allelic peptide profiling is shown in FIG. 9C. LC-MS/MSanalysis of HLA-associated peptides revealed characteristics of class Iand class II HLA-associated peptides (FIGS. 10A and 10B). Sequence logorepresentations of isolated and sequenced class I HLA-A*02:01-associatedpeptides and class II HLA-DRβ*11:01-associated peptides are shown FIG.10A. The length distribution comparisons of both class IHLA-A*02:01-associated peptides (red) and class IIHLA-DRβ*11:01-associated peptides (blue) showed that both class I andclass II HLA-associated peptides followed the expected trends (FIG.10B).

70 HLA class I alleles and 47 HLA class II alleles were assessed formono-allelic approach as described herein. 70 unique HLA class I alleleswith affinity tags (Table. 1A) and 47 unique HLA class II alleles withaffinity tags (Table. 2A) were generated. Table. 1B shows the details of96 unique experiments using the 70 unique HLA class I alleles (in somecases the same allele was placed into multiple cell lines). Table. 2Bshows the details of 54 unique experiments performed using the 47 uniqueHLA class II alleles (in some cases the same allele was placed intomultiple cell lines).

TABLE 1A 70 Unique HLA Class I alleles # Unique Class I Alleles 1 A*02012 A*0202 3 A*0203 4 A*0206 5 A*0207 6 A*1101 7 A*2301 8 A*2501 9 A*260110 A*3001 11 A*3002 12 A*3101 13 A*3201 14 A*3301 15 A*3303 16 A*3402 17A*3601 18 A*6801 19 A*7401 20 B*0702 21 B*0801 22 B*1302 23 B*1401 24B*1402 25 B*1501 26 B*1502 27 B*1503 28 B*1509 29 B*1510 30 B*1801 31B*270502 32 B*3502 33 B*3503 34 B*3701 35 B*3801 36 B*3802 37 B*3901 38B*3906 39 B*4001 40 B*4002 41 B*4006 42 B*4101 43 B*4201 44 B*4501 45B*4601 46 B*4801 47 B*4901 48 B*5001 49 B*5201 50 B*5301 51 B*5401 52B*5501 53 B*5703 54 B*5801 55 B*5802 56 B*8101 57 C*0102 58 C*0202 59C*0303 60 C*0401 61 C*0602 62 C*0701 63 C*0702 64 C*0704 65 C*1203 66C*1701 67 C*1801 68 F*0101 69 G*0101 70 E*0101

TABLE 1B Libraries and Cell Lines for HLA class I alleles # UniqueExperiments Wave of Class I # Library Allele Cell Line 1 W1 A0201 Hek2932 W1 A0201 HeLa 3 W1 A0201 A375 4 W1 A0201 expi293 5 W1 A1101 expi293 6W1 A1101 HeLa 7 W1 A2301 Hek293 8 W1 A2301 A375 9 W1 A2301 expi293 10 W1A2601 A375 11 W1 A2601 HeLa 12 W1 A2601 expi293 13 W1 A3201 A375 14 W1A3201 HeLa 15 W1 A3201 expi293 16 W1 A3601 expi293 17 W1 B0702 expi29318 W1 B0702 HeLa 19 W1 B0801 expi293 20 W1 B0801 HeLa 21 W1 B0801 A37522 W1 B1402 Hek293 23 W1 B1402 A375 24 W1 B1402 expi293 25 W1 B1402 HeLa26 W1 B1501 expi293 27 W1 B1509 HeLa 28 W1 B1509 expi293 29 W1 B1801HeLa 30 W1 B1801 expi293 31 W1  B270502 HeLa 32 W1  B270502 expi293 33W1 B4001 HeLa 34 W1 B4001 expi293 35 W2 A2501 expi293 36 W2 A3001expi293 37 W2 A3002 expi293 38 W2 A3101 expi293 39 W2 A3303 expi293 40W2 A6801 expi293 41 W2 A7401 expi293 42 W2 B1302 expi293 43 W2 B1503expi293 44 W2 B3503 expi293 45 W2 B3701 expi293 46 W2 B3801 expi293 47W2 B3901 expi293 48 W2 B4002 expi293 49 W2 B4101 expi293 50 W2 B4201expi293 51 W2 B4501 expi293 52 W2 B4601 expi293 53 W2 B4801 expi293 54W2 B4901 expi293 55 W2 B5001 expi293 56 W2 B5201 expi293 57 W2 B5301expi293 58 W2 B5501 expi293 59 W2 B5501 A375 60 W2 B5703 expi293 61 W2B5801 expi293 62 W2 B5801 A375 63 W2 B5802 expi293 64 W2 B8101 expi29365 W2 C0401 expi293 66 W2 C0401 A375 67 W2 C0602 expi293 68 W2 C0602A375 69 W2 C0701 expi293 70 W2 C0701 A375 71 W2 C0702 expi293 72 W2C0702 A375 73 W3 A3301 expi293 74 W3 A0206 expi293 75 W3 A0202 expi29376 W3 A3402 expi293 77 W3 A0207 expi293 78 W3 A0203 expi293 79 W3 B3502expi293 80 W3 B1401 expi293 81 W3 B3906 expi293 82 W3 B1510 expi293 83W3 B4006 expi293 84 W3 B1502 expi293 85 W3 B3802 expi293 86 W3 B5401expi293 87 W3 C0303 expi293 88 W3 C0202 expi293 89 W3 C1203 expi293 90W3 C0102 expi293 91 W3 C1701 expi293 92 W3 C0704 expi293 93 W3 C1801expi293 94 W2 E0101 expi293 95 W3 F0101 expi293 96 W3 G0101 expi293

TABLE 2A 47 Unique HLA Class II alleles # Unique Class II Alleles 1DPB1*0101 2 DPB1*0101 DPA*01:03 3 DPB1*0201 DPA*01:03 4 DPB1*0401DPA*01:03 5 DPB1*0402 DPA*01:03 6 DQ2*B0201*A0501 7 DQ2*B0202*A0201 8DQ6*B0602*A0102 9 DQ6*B1*0602 10 DRB1*0101 11 DRB1*0102 12 DRB1*0301 13DRB1*0302 14 DRB1*0401 15 DRB1*0402 16 DRB1*0403 17 DRB1*0404 18DRB1*0405 19 DRB1*0407 20 DRB1*0701 21 DRB1*0801 22 DRB1*0802 23DRB1*0803 24 DRB1*0804 25 DRB1*0901 26 DRB1*1001 27 DRB1*1101 28DRB1*1102 29 DRB1*1104 30 DRB1*1201 31 DRB1*1202 32 DRB1*1301 33DRB1*1302 34 DRB1*1303 35 DRB1*1401 36 DRB1*1501 37 DRB1*1502 38DRB1*1503 39 DRB1*1601 40 DRB3*0101 41 DRB3*0202 42 DRB3*0301 43DRB4*0103 44 DRB5*0101 45 HLA DM no affinity tag 46 HLA-DM with affinitytag 47 HLA-DO

TABLE 2B Libraries and Cell Lines for HLA class II alleles # UniqueExperiments Wave of Class II # Library Allele Cell Line 1 W1 DRB1_0101Hek293 2 W1 DRB1_0101 expi293 3 W1 DRB1_0102 Hek293 4 W1 DRB1_0102expi293 5 W1 DRB1_0701 Hek293 6 W1 DRB1_0701 expi293 7 W1 DRB1_1101Hek293 8 W1 DRB1_1101 HeLa 9 W1 DRB1_1101 A375 10 W1 DRB1_1101 expi29311 W1 DRB1_1501 Hek293 12 W1 DRB1_1501 expi293 13 W2 DRB1_0301 expi29314 W2 DRB1_0302 expi293 15 W2 DRB1_0401 expi293 16 W2 DRB1_0404 expi29317 W2 DRB1_0405 expi293 18 W2 DRB1_0407 expi293 19 W2 DRB1_0801 expi29320 W2 DRB1_0802 expi293 21 W2 DRB1_0804 expi293 22 W2 DRB1_0901 expi29323 W2 DRB1_1001 expi293 24 W2 DRB1_1104 expi293 25 W2 DRB1_1201 expi29326 W2 DRB1_1301 expi293 27 W2 DRB1_1302 expi293 28 W2 DRB1_1303 expi29329 W2 DRB1_1401 expi293 30 W2 DRB1_1502 expi293 31 W2 DRB1_1503 expi29332 W2 DRB1_1601 expi293 33 W2 DRB4_0103 expi293 34 W2 DPB1_0101 expi29335 W2 DPB1_0201 expi293 36 W2 DPB1_0401 expi293 37 W2 DPB1_0402 expi29338 W2 DQ2_B0201_A0501 expi293 39 W2 DQ2_B0202_A0201 expi293 40 W2DQ6_B0602_A0102 expi293 41 W2 HLA DM no tag expi293 42 W3 DRB1*0402expi293 43 W3 DRB1*0403 expi293 44 W3 DRB1*1102 expi293 45 W3 DRB1*1202expi293 46 W3 DRB1*0803 expi293 47 W3 DRB3*0101 expi293 48 W3 DRB3*0202expi293 49 W3 DRB5*0101 expi293 50 W3 DRB3*0301 expi293 Si W3 HLA-DOexpi293 52 W3 DPB1 0101 expi293 53 W3 DQ6 B1 0602 expi293 54 W3 HLA-DMtagged expi293

Example 4. Tandem Universal IP of Class II HLA Complexes with MultipleAffinity Tags

Class II HLA complexes are formed by α-chain and β-chain pairing, eachof which can be tagged with a different affinity tag. A serial IP usingboth affinity tags enables the deconvolution of α-chain and β-chainpairing and unambiguous peptide-binding assignments to class II HLAcomplexes. A schematic representation of class II HLA constructsengineered for expression by different cell types for Universal IPpipeline is shown in FIG. 11A. Schematic representations of the possibleclass II HLA complexes that can form upon expression of FIG. 11Aconstructs in cell lines expressing endogenous class II HLA α-chain andβ-chain subunits are shown in FIG. 11B.

Schematics of a serial Universal IP strategy that can be used fordeconvolution of α-chain and β-chain pairing and unambiguouspeptide-binding assignments to specific class II HLA complexes aredepicted in FIG. 12A. Cells expressing dual-affinity tagged class II HLAconstructs were lysed, biotinylated, and incubated with beads coupled toanti-HA antibodies. Class II HLA complexes with HA-tagged subunits wereisolated, washed, and eluted using an HA peptide (YPYDVPDYA). Theelution was then incubated with beads coupled to either NeutrAvidin orstreptavidin to isolate the HA-tagged and biotin-tagged class II HLAcomplexes. Peptides bound to dual-tagged class II HLA complexes are theneluted and sequenced by LC-MS/MS. A Western blot and loading control(Ponceau S stained gel) demonstrated the specificity of the serialUniversal IP pipeline. A Western blot validated the serial Universal IPstrategy in HEK293T expressing dual-tagged HLA-DRB*11:01 constructs(FIG. 12B). An anti-HA antibody was used to follow the serial enrichmentprocess. A Ponceau S stained gel was used as a Western blot loadingcontrol. A Western blot of a negative control experiment where cellsexpressing dual-affinity tagged class II HLA construct HLA-DRB*11:01were lysed and incubated with beads coupled to anti-HA antibodieswithout biotinylation is shown in FIG. 12C. As shown in FIG. 12C, noenrichment was observed when the biotinylation step was removed from theserial Universal IP protocol.

Example 5. A Mono-Allelic HLA-Peptidome Profiling Approach thatImplements a Biotin Affinity Tag

A schematic representation of a mono-allelic HLA-peptidome profilingapproach that implements a biotin affinity tag is shown in FIG. 14A andFIG. 14B. An exemplary embodiment of the present disclosure makes use ofthe biotin acceptor peptide (BAP) that is biotinylated on a lysine (K)residue by a BirA enzyme. The BAP peptide sequence contains a lysineresidue that is biotinylated upon the addition of BirA enzyme, biotin,and ATP. The biotinylated product displays high affinity forstreptavidin/NeutrAvidin. Streptavidin/NeutrAvidin beads can be used toenrich for the biotinylated BAP peptide sequence.

Example 6. Targeted Epitope Discovery Platform

A cell line of interest (e.g., 2HEK293T, expi293, HeLa, A375, 721.221,JEG-3, K562, Jurkat, Hep G2, SH-SY5Y, CACO-2, U937, U-2 OS, ExpiCHO, CHOor THP1) or primary cells (e.g., cells from a subject with a disease orcondition) can be transfected/transduced with a class I or II HLAconstruct containing a tag (e.g., BAP sequence) on the N- or C-terminuswith or without selection to enrich for HLA expressing cells (FIG. 15).The cells can then be transfected or transduced with a second plasmidthat contains an epitope fragment or a chain of epitopes that can beexpressed and presented on the tag-labeled HLA molecule. Alternatively,both the HLA allele plasmid and the epitope plasmid can be co-deliveredinto the cells followed by expansion and/or selection. These engineeredcells are then lysed, biotinylated, and the HLA molecule is enrichedfrom the lysate (e.g., using streptavidin beads). The peptides areeluted from the HLA molecule and analyzed, e.g., by LC-MS/MS. Thismethod permits analysis of how epitopes are processed and presented bydifferent alleles. This method can also be utilized to improve epitopedelivery and design.

Example 7. Allele Multiplexing

A DNA construct can be designed to express multiple class I heavy chainsor multiple class II < or ® chains that contain one or more tags (FIG.16). Each HLA construct can be expressed from the same gene constructthat includes a ribosomal skipping sequence (F2A, T2A, P2A, etc.) or anIRES element. A desired cell line can be transduced or transfected withthis plasmid to induce expression of multiple HLA alleles that aretagged and subsequently enriched. Alternatively, a cell line can betransduced or transfected with multiple plasmids that each contain asingle HLA allele. The peptides bound to the HLA alleles can then beanalyzed, e.g., by LC-MS/MS. This platform permits generation of celllines with multiple alleles. This can be used, for example, to match apatient's HLA-type. This will permit generation of peptide epitopepatterns for different allele combinations.

Example 8. Improved Prediction of Processing and Allele-Specific Binding

NetMHC is an allele-specific method which trains a separate predictorfor each allele's binding dataset, and NetMHCpan is pan-allele methodwhose inputs are vector encodings of both a peptide and a subsequence ofa particular MHC molecule. The conventional wisdom is that NetMHCperforms better on alleles with many assayed ligands, whereas NetMHCpanperforms better for less well-characterized alleles. However, it hasbeen shown that NetMHCpan is not accurate when no relevant data wasincluded in the training sets.

Mono-allelic approach as described herein (FIG. 21) uncoveredHLA-binding peptides that were poorly scored by NetMHCpan butbiochemically validated as strong binders. FIG. 20A shows exemplary HLAbinding peptides for A*01:01, B*51:01, A*29:02, and B*54:01 allelesuncovered using the presently described mono-allelic approach. FIG. 20Bshows the rates of incorrect assignment in 100 simulated deconvolutions.A random six allele patient HLA genotype (2 alleles each of HLA-A,HLA-B, and HLA-C, sampling at US allele frequencies) was generated. Foreach allele, 500 peptides from relevant mono-allelic experiment weresampled and combined to create mock 3000 peptide multi-allelic data set.Each peptide was assigned to allele that yields the best NetMHCpan %rank score to determine percentage of peptides incorrectly assigned byNetMHCpan. This process was repeated 100 times. As shown in FIG. 22,both processing and allele-specific binding prediction weresignificantly improved.

Paragraphs of the Disclosure

Provided herein is a method of characterizing HLA-peptide complexescomprising: providing a population of cells, wherein one or more cellsof the population of cells comprise a polynucleic acid comprising asequence encoding an affinity acceptor tagged class I or class II HLAallele, wherein the sequence encoding an affinity acceptor tagged HLAcomprises a sequence encoding a recombinant class I or class II HLAallele operatively linked to a sequence encoding an affinity acceptorpeptide; expressing the affinity acceptor tagged HLA in at least onecell of the one or more cells of the population of cells, therebyforming affinity acceptor tagged HLA-peptide complexes in the at leastone cell; enriching for the affinity acceptor tagged HLA-peptidecomplexes; and characterizing HLA-peptide complexes. In someembodiments, the encoded affinity acceptor tagged class I or class IIHLA allele is a soluble affinity acceptor tagged class I or class II HLAallele.

In some embodiments, the characterizing comprises characterizing apeptide bound to the affinity acceptor tagged HLA-peptide complex fromthe enriching. In some embodiments, the method comprises carrying outthe steps of the method for two or more class I and/or class II HLAalleles. In some embodiments, the two or more class I and/or class IIHLA alleles comprise at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50 class I and/or classII HLA alleles. In some embodiments, the affinity acceptor taggedHLA-peptide complexes comprise a transmembrane domain. In someembodiments, the affinity acceptor tagged HLA-peptide complexes comprisean intracellular domain. In some embodiments, the affinity acceptortagged HLA-peptide complexes are not secreted. In some embodiments, theaffinity acceptor tagged HLA-peptide complexes incorporate into a cellmembrane when expressed. In some embodiments, the affinity acceptortagged HLA-peptide complexes are soluble affinity acceptor taggedHLA-peptide complexes. In some embodiments, the affinity acceptor taggedHLA-peptide complexes are not soluble affinity acceptor taggedHLA-peptide complexes. In some embodiments, the method further comprisesgenerating an HLA-allele specific peptide database. In some embodiments,the recombinant class I or class II HLA allele is a single recombinantclass I or class II HLA allele.

In some embodiments, the method comprises: providing a population ofcells each comprising one or more cells comprising an affinity acceptortagged HLA, wherein the affinity acceptor tagged HLA comprises adifferent recombinant polypeptide encoded by a different HLA alleleoperatively linked to an affinity acceptor peptide; enriching foraffinity acceptor tagged HLA-peptide complexes; and characterizing apeptide or a portion thereof bound to the affinity acceptor taggedHLA-peptide complex from the enriching.

In some embodiments, the method comprises introducing one or morepeptides to the population of cells. In some embodiments, theintroducing comprises contacting the population of cells with the one ormore peptides or expressing the one or more peptides in the populationof cells. In some embodiments, the introducing comprises contacting thepopulation of cells with one or more nucleic acids encoding the one ormore peptides. In some embodiments, the one or more nucleic acidsencoding the one or more peptides is DNA. In some embodiments, the oneor more nucleic acids encoding the one or more peptides is RNA,optionally wherein the RNA is mRNA. In some embodiments, the enrichingdoes not comprise use of a tetramer reagent.

In some embodiments, the characterizing comprises determining thesequence of a peptide or a portion thereof bound to the affinityacceptor tagged HLA-peptide complex from the enriching, optionallydetermining whether a peptide or a portion thereof is modified. In someembodiments, the determining comprises biochemical analysis, massspectrometry analysis, MS analysis, MS/MS analysis, LC-MS/MS analysis,or a combination thereof. In some embodiments, the characterizingcomprises evaluating a binding affinity or stability of a peptide or aportion thereof bound to the affinity acceptor tagged HLA-peptidecomplex from the enriching. In some embodiments, the characterizingcomprises determining whether a peptide or a portion thereof bound tothe affinity acceptor tagged HLA-peptide complex from the enrichingcontains one or more mutations. In some embodiments, the characterizingcomprises evaluating associations of peptides with HLA molecules in theaffinity acceptor tagged HLA-peptide complexes.

In some embodiments, the method comprises expressing a library ofpeptides in the population of cells, thereby forming a library ofaffinity acceptor tagged HLA-peptide complexes. In some embodiments, themethod comprises contacting to the population of cells a library ofpeptides or a library of sequences encoding peptides, thereby forming alibrary of affinity acceptor tagged HLA-peptide complexes. In someembodiments, the library comprises a library of peptides associated witha disease or condition. In some embodiments, the library comprises alibrary of peptides derived from a polypeptide drug, such as a biologic(e.g., an antibody drug).

In some embodiments, the disease or condition is cancer, an infectionwith an infectious agent, or an autoimmune reaction. In someembodiments, the method comprises introducing the infectious agent orportions thereof into one or more cells of the population of cells. Insome embodiments, the method comprises introducing a polypeptide drug,such as a biologic (e.g., an antibody drug) or portions thereof into oneor more cells of the population of cells. In some embodiments, themethod comprises characterizing one or more peptides from theHLA-peptide complexes, optionally wherein the peptides are from one ormore target proteins of the infectious agent or the polypeptide drug. Insome embodiments, the method comprises characterizing one or moreregions of the peptides from the one or more target proteins of theinfectious agent or the polypeptide drug.

In some embodiments, the method comprises identifying peptides from theHLA-peptide complexes derived from an infectious agent. In someembodiments, the population of cells is from a biological sample from asubject with a disease or condition. In some embodiments, the populationof cells is a cell line. In some embodiments, the population of cells isa population of primary cells. In some embodiments, the recombinantclass I or class II HLA allele is matched to a subject with a disease orcondition.

In some embodiments, the peptide from the affinity acceptor taggedHLA-peptide complex is capable of activating a T cell from a subjectwhen presented by an antigen presenting cell. In some embodiments, thecharacterizing comprises comparing HLA-peptide complexes from cancercells to HLA-peptide complexes from non-cancer cells. In someembodiments, the population of cells comprises a plurality ofpopulations of cells, each population of cells expressing a differentrecombinant class I or class II HLA allele. In some embodiments, eachpopulation of cells of the plurality is in a same or a separatecontainer.

In some embodiments, the method further comprises isolating peptidesfrom the affinity acceptor tagged HLA-peptide complexes before thecharacterizing. In some embodiments, an HLA-peptide complex is isolatedusing an anti-HLA antibody. In some cases, an HLA-peptide complex withor without an affinity tag is isolated using an anti-HLA antibody. Insome cases, a soluble HLA (sHLA) with or without an affinity tag isisolated from media of a cell culture. In some cases, a soluble HLA(sHLA) with or without an affinity tag is isolated using an anti-HLAantibody. For example, an HLA, such as a soluble HLA (sHLA) with orwithout an affinity tag, can be isolated using a bead or columncontaining an anti-HLA antibody. In some embodiments, the peptides areisolated using anti-HLA antibodies. In some cases, a soluble HLA (sHLA)with or without an affinity tag is isolated using an anti-HLA antibody.In some cases, a soluble HLA (sHLA) with or without an affinity tag isisolated using a column containing an anti-HLA antibody. In someembodiments, the method further comprises removing one or more aminoacids from a terminus of a peptide bound to an affinity acceptor taggedHLA-peptide complex.

In some embodiments, the population of cells is a population of low cellsurface HLA class I or class II expressing cells. In some embodiments,the population of cells expresses one or more endogenous HLA alleles. Insome embodiments, the population of cells is an engineered population ofcells lacking one or more endogenous HLA class I alleles. In someembodiments, the population of cells is an engineered population ofcells lacking endogenous HLA class I alleles. In some embodiments, thepopulation of cells is an engineered population of cells lacking one ormore endogenous HLA class II alleles. In some embodiments, thepopulation of cells is an engineered population of cells lackingendogenous HLA class II alleles. In some embodiments, the population ofcells is an engineered population of cells lacking endogenous HLA classI alleles and endogenous HLA class II alleles. In some embodiments, thepopulation of cells is a knock-out of one or more HLA class I alleles.In some embodiments, the population of cells is a knock-out of one ormore HLA class II alleles. In some embodiments, the population of cellsis a knock-out of all HLA class I alleles. In some embodiments, thepopulation of cells is a knock-out of all HLA class II alleles. In someembodiments, the population of cells is a knock-out of all HLA class Ialleles and a knock-out of all HLA class II alleles. In someembodiments, the sequence encoding the recombinant class I or class IIHLA allele encodes a class I HLA. In some embodiments, the class I HLAis selected from the group consisting of HLA-A, HLA-B, HLA-C, HLA-E,HLA-F, and HLA-G. In some embodiments, the sequence encoding therecombinant class I or class II HLA allele encodes a class II HLA. Insome embodiments, the class II HLA is selected from the group consistingof HLA-DR, HLA-DQ, and HLA-DP. In some embodiments, the class II HLAcomprises a HLA class II α-chain, a HLA class II β-chain, or acombination thereof. In some embodiments, each sequence encodes at leasttwo different class I and/or class II HLA alleles.

In some embodiments, the at least two different class I and/or class IIHLA alleles are each operatively linked to a sequence encoding anaffinity acceptor peptide. In some embodiments, the at least twodifferent class I and/or class II HLA alleles are each operativelylinked to a sequence encoding a different affinity acceptor peptide. Insome embodiments, the at least two different class I and/or class II HLAalleles are each operatively linked to a sequence encoding an affinityacceptor peptide. In some embodiments, one or more of the at least twodifferent class I and/or class II HLA alleles is operatively linked to asequence encoding a first affinity acceptor peptide and one or more ofthe at least two different class I and/or class II HLA alleles isoperatively linked to a sequence encoding a second affinity acceptorpeptide. In some embodiments, the at least two different class I and/orclass II HLA alleles are each operatively linked to a sequence encodinga different affinity acceptor peptide. In some embodiments, each of theat least two different class I and/or class II HLA alleles are eachoperatively linked to a sequence encoding a different affinity acceptorpeptide. In some embodiments, the at least two different class I and/orclass II HLA alleles are each operatively linked to a sequence encodingan affinity tag. In some embodiments, the method comprises administeringat least a second polynucleic acid comprising a sequence encoding adifferent recombinant HLA allele operatively linked to the same or adifferent affinity acceptor peptide.

In some embodiments, the sequence encoding the affinity acceptor peptideis operatively linked to a sequence that encodes an extracellularportion of the recombinant class I or class II HLA allele. In someembodiments, the encoded affinity acceptor peptide is expressedextracellularly. In some embodiments, the encoded affinity acceptorpeptide is located on an extracellular site of the recombinant class Ior class II HLA allele. In some embodiments, the sequence encoding theaffinity acceptor peptide is operatively linked to the N-terminus of thesequence encoding the recombinant class I or class II HLA allele. Insome embodiments, the sequence encoding the affinity acceptor peptide isoperatively linked to a sequence that encodes an intracellular portionof the recombinant class I or class II HLA allele. In some embodiments,the encoded affinity acceptor peptide is expressed intracellularly. Insome embodiments, the sequence encoding the affinity acceptor peptide isoperatively linked to the C-terminus of the sequence encoding therecombinant class I or class II HLA allele. In some embodiments, thesequence encoding the affinity acceptor peptide is operatively linked toan internal sequence of the sequence encoding the recombinant class I orclass II HLA allele, such as a flexible loop sequence. In someembodiments, the sequence encoding the affinity acceptor peptide isoperatively linked to the sequence encoding the recombinant class I orclass II HLA allele by a linker. In some embodiments, enrichingcomprises enriching for intact cells expressing the affinity acceptortagged HLA-peptide complexes. In some embodiments, the method does notcomprise lysing the cells before enriching. In some embodiments, themethod further comprises lysing the one or more cells before enriching.In some embodiments, enriching comprises contacting an affinity acceptorpeptide binding molecule to the affinity acceptor tagged HLA-peptidecomplexes, wherein the affinity acceptor peptide binding molecule bindsspecifically to the affinity acceptor peptide.

In some embodiments, the affinity acceptor peptide comprises a tagsequence comprising a biotin acceptor peptide (BAP), poly-histidine tag,poly-histidine-glycine tag, poly-arginine tag, poly-aspartate tag,poly-cysteine tag, poly-phenylalanine, c-myc tag, Herpes simplex virusglycoprotein D (gD) tag, FLAG tag, KT3 epitope tag, tubulin epitope tag,T7 gene 10 protein peptide tag, streptavidin tag, streptavidin bindingpeptide (SPB) tag, Strep-tag, Strep-tag II, albumin-binding protein(ABP) tag, alkaline phosphatase (AP) tag, bluetongue virus tag (B-tag),calmodulin binding peptide (CBP) tag, chloramphenicol acetyl transferase(CAT) tag, choline-binding domain (CBD) tag, chitin binding domain (CBD)tag, cellulose binding domain (CBP) tag, dihydrofolate reductase (DHFR)tag, galactose-binding protein (GBP) tag, maltose binding protein (MBP),glutathione-S-transferase (GST), Glu-Glu (EE) tag, human influenzahemagglutinin (HA) tag, horseradish peroxidase (HRP) tag, NE-tag, HSVtag, ketosteroid isomerase (KSI) tag, KT3 tag, LacZ tag, luciferase tag,NusA tag, PDZ domain tag, AviTag, Calmodulin-tag, E-tag, S-tag, SBP-tag,Softag 1, Softag 3, TC tag, VSV-tag, Xpress tag, Isopeptag, SpyTag,SnoopTag, Profinity eXact tag, Protein C tag, S1-tag, S-tag,biotin-carboxy carrier protein (BCCP) tag, green fluorescent protein(GFP) tag, small ubiquitin-like modifier (SUMO) tag, tandem affinitypurification (TAP) tag, HaloTag, Nus-tag, Thioredoxin-tag, Fc-tag, CYDtag, HPC tag, TrpE tag, ubiquitin tag, VSV-G epitope tag, V5 tag,sortase tag, a tag the forms a covalent peptide bond to a bead, or acombination thereof; optionally, wherein the affinity acceptor peptidecomprises two or more repeats of a tag sequence.

In some embodiments, the affinity acceptor peptide binding molecule isbiotin or an antibody specific to the affinity acceptor peptide. In someembodiments, the enriching comprises contacting an affinity molecule tothe affinity acceptor tagged HLA-peptide complexes, wherein the affinitymolecule binds specifically to the affinity acceptor peptide bindingmolecule.

In some embodiments, the affinity molecule comprises a molecule thatbinds to biotin. For example, the affinity molecule can comprisestreptavidin, NeutrAvidin, including protein homologs from otherorganisms and derivatives thereof.

In some embodiments, enriching comprises immunoprecipitating affinityacceptor tagged HLA-peptide complexes. In some embodiments, the affinityacceptor peptide binding molecule is attached to a solid surface. Insome embodiments, the affinity molecule is attached to a solid surface.In some embodiments, the solid surface is a bead. In some embodiments,enriching comprises immunoprecipitating affinity acceptor taggedHLA-peptide complexes with an affinity acceptor peptide binding moleculethat binds specifically to the affinity acceptor peptide.

In some embodiments, the affinity acceptor peptide binding molecule doesnot specifically interact with the amino acid sequence of the encodedrecombinant class I or class II HLA. In some embodiments, enrichingcomprises contacting an affinity molecule specific to an extracellularportion of the recombinant class I or class II HLA allele. In someembodiments, enriching comprises contacting an affinity moleculespecific to an N-terminal portion of the recombinant class I or class IIHLA allele.

In some embodiments, providing comprises contacting the population ofcells with the polynucleic acid. In some embodiments, contactingcomprises transfecting or transducing. In some embodiments, providingcomprises contacting the population of cells with a vector comprisingthe polynucleic acid. In some embodiments, the vector is a viral vector.In some embodiments, the polynucleic acid is stably integrated into thegenome of the population of cells.

In some embodiments, the sequence encoding the recombinant class I orclass II HLA comprises a sequence encoding a HLA class I α-chain. Insome embodiments, the method further comprises expressing a sequenceencoding β2 microglobulin in the one or more cells. In some embodiments,the sequence encoding β2 microglobulin is connected to the sequenceencoding the HLA class I α-chain. In some embodiments, the sequenceencoding β2 microglobulin is connected to the sequence encoding the HLAclass I α-chain by a linker. In some embodiments, the sequence encodingβ2 microglobulin is connected to a sequence encoding a second affinityacceptor peptide. In some embodiments, the sequence encoding therecombinant class I or class II HLA comprises a sequence encoding a HLAclass II α-chain. In some embodiments, the method further comprisesexpressing a sequence encoding a HLA class II β-chain in the one or morecells. In some embodiments, the sequence encoding the HLA class IIβ-chain is connected to the sequence encoding the HLA class II α-chain.In some embodiments, the sequence encoding the HLA class II β-chain isconnected to the sequence encoding the HLA class II α-chain by a linker.In some embodiments, the sequence encoding the HLA class II β-chain isconnected to a sequence encoding a second affinity acceptor peptide.

In some embodiments, the second affinity acceptor peptide is differentthan the first affinity acceptor peptide and is selected from the groupconsisting of biotin acceptor peptide (BAP), poly-histidine tag,poly-histidine-glycine tag, poly-arginine tag, poly-aspartate tag,poly-cysteine tag, poly-phenylalanine, c-myc tag, Herpes simplex virusglycoprotein D (gD) tag, FLAG tag, KT3 epitope tag, tubulin epitope tag,T7 gene 10 protein peptide tag, streptavidin tag, streptavidin bindingpeptide (SPB) tag, Strep-tag, Strep-tag II, albumin-binding protein(ABP) tag, alkaline phosphatase (AP) tag, bluetongue virus tag (B-tag),calmodulin binding peptide (CBP) tag, chloramphenicol acetyl transferase(CAT) tag, choline-binding domain (CBD) tag, chitin binding domain (CBD)tag, cellulose binding domain (CBP) tag, dihydrofolate reductase (DHFR)tag, galactose-binding protein (GBP) tag, maltose binding protein (MBP),glutathione-S-transferase (GST), Glu-Glu (EE) tag, human influenzahemagglutinin (HA) tag, horseradish peroxidase (HRP) tag, NE-tag, HSVtag, ketosteroid isomerase (KSI) tag, KT3 tag, LacZ tag, luciferase tag,NusA tag, PDZ domain tag, AviTag, Calmodulin-tag, E-tag, S-tag, SBP-tag,Softag 1, Softag 3, TC tag, VSV-tag, Xpress tag, Isopeptag, SpyTag,SnoopTag, Profinity eXact tag, Protein C tag, S1-tag, S-tag,biotin-carboxy carrier protein (BCCP) tag, green fluorescent protein(GFP) tag, small ubiquitin-like modifier (SUMO) tag, tandem affinitypurification (TAP) tag, HaloTag, Nus-tag, Thioredoxin-tag, Fc-tag, CYDtag, HPC tag, TrpE tag, ubiquitin tag, VSV-G epitope tag, V5 tag, and acombination thereof; optionally, wherein the first or second affinityacceptor peptide comprises two or more repeats of a tag sequence.

In some embodiments, the linker comprises a polynucleic acid sequenceencoding a cleavable linker. In some embodiments, the cleavable linkeris a ribosomal skipping site or an internal ribosomal entry site (IRES)element. In some embodiments, the ribosomal skipping site or IRES iscleaved when expressed in the cells. In some embodiments, the ribosomalskipping site is selected from the group consisting of F2A, T2A, P2A,and E2A. In some embodiments, the IRES element is selected from commoncellular or viral IRES sequences.

In some embodiments, the determining comprises performing biochemicalanalysis or mass spectrometry, such as tandem mass spectrometry. In someembodiments, the determining comprises obtaining a peptide sequence thatcorresponds to an MS/MS spectra of one or more peptides isolated fromthe enriched affinity acceptor tagged HLA-peptide complexes from apeptide database; wherein one or more sequences obtained identifies thesequence of the one or more peptides. In some embodiments, the peptidedatabase is a no-enzyme specificity peptide database, such as a withoutmodification database or a with modification database. In someembodiments, the method further comprises searching the peptide databaseusing a reversed-database search strategy.

In some embodiments, the population of cells is a cell line. In someembodiments, the population of cells is a human cell line. In someembodiments, the population of cells is a mouse cell line. In someembodiments, the population of cells is a CHO cell line. In someembodiments, the population of cells is a cell line selected fromHEK293T, expi293, HeLa, A375, 721.221, JEG-3, K562, Jurkat, and THP1. Insome embodiments, the population of cells is treated with one or morecytokines, checkpoint inhibitors, epigenetically-active drugs, IFN-γ,agents that alter antigen processing (such as peptidase inhibitors,proteasome inhibitors, and TAP inhibitors), or a combination thereof. Insome embodiments, the population of cells is treated with one or morereagents that modulate a metabolic pathway or a metabolic status of thecells. In some embodiments, the population of cells is treated with oneor more reagents that modulate the cellular proteome of the cells. Insome embodiments, the population of cells is treated with one or morereagents that modulate or regulate cellular expression or transcription(e.g. AIRE or a CREB binding protein or modulators thereof) of thecells. In some embodiments, the population of cells is treated with oneor more reagents that modulate or regulate a transcription factor of thecells. In some embodiments, the population of cells is treated with oneor more reagents that modulate or regulate cellular expression ortranscription of an HLA of the cells. In some embodiments, thepopulation of cells is treated with one or more reagents that modulateor regulate cellular expression or transcription of the proteome of thecells.

In some embodiments, the population of cells comprises at least 10⁵cells, at least 10⁶ cells or at least 10⁷ cells. In some embodiments,the population of cells is a population of dendritic cells, macrophages,cancer cells or B-cells. In some embodiments, the population of cellscomprises tumor cells. In some embodiments, the population of cells iscontacted with an agent prior to isolating said HLA-peptide complexesfrom the one or more cells. In some embodiments, said agent is aninflammatory cytokine, a chemical agent, an adjuvant, a therapeuticagent or radiation.

In some embodiments, the HLA allele is a mutated HLA allele. In someembodiments, the sequence encoding the HLA allele comprises a barcodesequence. In some embodiments, the method further comprises assaying forexpression of the affinity acceptor tagged class I or class II HLAallele. In some embodiments, the assaying comprises assaying comprisessequencing an affinity acceptor tagged class I or class II HLA allele,detecting affinity acceptor tagged class I or class II HLA allele RNA,detecting affinity acceptor tagged class I or class II HLA alleleprotein, or a combination thereof. In some embodiments, assaying forexpression can comprise a Western blot assay, fluorescent activated cellsorting (FACS), mass spectrometry (MS), a microarray hybridizationassay, an RNA-seq assay, a polymerase chain reaction assay, a LAMPassay, a ligase chain reaction assay, a Southern blot assay, a Northernblot assay, or an enzyme-linked immunosorbent assay (ELISA).

In some embodiments, the method comprises carrying out the steps of themethod for different HLA alleles. In some embodiments, each differentHLA allele comprises a unique barcode sequence. In some embodiments,each polynucleic acid encoding a different HLA allele comprises a uniquebarcode sequence.

Provided herein is a HLA-allele specific binding peptide sequencedatabase obtained by carrying out a method described herein. Providedherein is a combination of two or more HLA-allele specific bindingpeptide sequence databases obtained by carrying out a method describedherein repeatedly, each time using a different HLA-allele. Providedherein is a method for generating a prediction algorithm for identifyingHLA-allele specific binding peptides, comprising training a machine witha peptide sequence database described herein or a combination describedherein.

In some embodiments, the machine combines one or more linear models,support vector machines, decision trees and neural networks. In someembodiments, a variable used to train the machine comprises one or morevariables selected from the group consisting of peptide sequence, aminoacid physical properties, peptide physical properties, expression levelof the source protein of a peptide within a cell, protein stability,protein translation rate, ubiquitination sites, protein degradationrate, translational efficiencies from ribosomal profiling, proteincleavability, protein localization, motifs of host protein thatfacilitate TAP transport, host protein is subject to autophagy, motifsthat favor ribosomal stalling, and protein features that favor NMD.

In some embodiments, the motifs that favor ribosomal stalling comprisepolyproline or polylysine stretches. In some embodiments, the proteinfeatures that favor NMD are selected from the group consisting of a long3′ UTR, a stop codon greater than 50nt upstream of last exon:exonjunction, and peptide cleavability.

Provided herein is a method for identifying HLA-allele specific bindingpeptides comprising analyzing the sequence of a peptide with a machinewhich has been trained with a peptide sequence database obtained bycarrying out a method described herein for the HLA-allele. In someembodiments, the method comprises determining the expression level ofthe source protein of the peptide within a cell; and wherein the sourceprotein expression is a predictive variable used by the machine. In someembodiments, the expression level is determined by measuring the amountof source protein or the amount of RNA encoding said source protein.

Provided herein is a composition comprising a recombinant polynucleicacid comprising two or more sequences each encoding an affinity acceptortagged HLA, wherein the sequences encoding the affinity acceptor taggedHLAs comprise a sequence encoding a different recombinant HLA class Iα-chain allele, a sequence encoding an affinity acceptor peptide, andoptionally, a sequence encoding β2 microglobulin; wherein the sequencesof (a) and (b), and optionally (c), are operatively linked.

Provided herein is a composition comprising a recombinant polynucleicacid comprising two or more sequences each comprising a sequenceencoding an affinity acceptor tagged HLA, wherein the sequences encodingthe affinity acceptor tagged HLAs comprise a sequence encoding arecombinant HLA class II α-chain allele, a sequence encoding an affinityacceptor peptide, and optionally, a sequence encoding a HLA class IIβ-chain; wherein the sequences of (a) and (b), and optionally (c), areoperatively linked. In some embodiments, the recombinant polynucleicacid is isolated. In some embodiments, the class I HLA is selected fromthe group consisting of HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, and HLA-G. Insome embodiments, the class II HLA is selected from the group consistingof HLA-DR, HLA-DQ, and HLA-DP.

In some embodiments, the sequence encoding the affinity acceptor peptideis operatively linked to a sequence that encodes for an extracellularportion of the recombinant HLA allele. In some embodiments, the sequenceencoding the affinity acceptor molecule is operatively linked to theN-terminus of the sequence encoding the recombinant HLA allele. In someembodiments, the sequence encoding the affinity acceptor peptide isoperatively linked to a sequence encoding an intracellular portion ofthe recombinant HLA allele. In some embodiments, the sequence encodingthe affinity acceptor peptide is operatively linked to the C-terminus ofthe sequence encoding the recombinant HLA allele. In some embodiments,the sequence encoding the affinity acceptor peptide is operativelylinked to the sequence encoding the recombinant HLA allele by a linker.

In some embodiments, the two or more sequences encoding an affinityacceptor tagged HLA are expressed from the same polynucleotide. In someembodiments, the two or more sequences encoding an affinity acceptortagged HLA are expressed from different polynucleotides. In someembodiments, the encoded affinity acceptor peptide binds specifically toan affinity acceptor peptide binding molecule. In some embodiments, thetwo or more sequences encoding an affinity acceptor tagged HLA comprisetwo or more affinity acceptor peptides. In some embodiments, the two ormore sequences encoding an affinity acceptor tagged HLA comprise threeor more sequences encoding an affinity acceptor tagged HLA, wherein atleast two of the three or more sequences encoding an affinity acceptortagged HLA comprises the same affinity acceptor peptide. In someembodiments, the two or more affinity acceptor peptides are unique foreach of the two or more sequences encoding an affinity acceptor taggedHLA.

In some embodiments, the encoded affinity acceptor peptide is selectedfrom the group consisting of biotin acceptor peptide (BAP),poly-histidine tag, poly-histidine-glycine tag, poly-arginine tag,poly-aspartate tag, poly-cysteine tag, poly-phenylalanine, c-myc tag,Herpes simplex virus glycoprotein D (gD) tag, FLAG tag, KT3 epitope tag,tubulin epitope tag, T7 gene 10 protein peptide tag, streptavidin tag,streptavidin binding peptide (SPB) tag, Strep-tag, Strep-tag II,albumin-binding protein (ABP) tag, alkaline phosphatase (AP) tag,bluetongue virus tag (B-tag), calmodulin binding peptide (CBP) tag,chloramphenicol acetyl transferase (CAT) tag, choline-binding domain(CBD) tag, chitin binding domain (CBD) tag, cellulose binding domain(CBP) tag, dihydrofolate reductase (DHFR) tag, galactose-binding protein(GBP) tag, maltose binding protein (MBP), glutathione-S-transferase(GST), Glu-Glu (EE) tag, human influenza hemagglutinin (HA) tag,horseradish peroxidase (HRP) tag, NE-tag, HSV tag, ketosteroid isomerase(KSI) tag, KT3 tag, LacZ tag, luciferase tag, NusA tag, PDZ domain tag,AviTag, Calmodulin-tag, E-tag, S-tag, SBP-tag, Softag 1, Softag 3, TCtag, VSV-tag, Xpress tag, Isopeptag, SpyTag, SnoopTag, Profinity eXacttag, Protein C tag, S1-tag, S-tag, biotin-carboxy carrier protein (BCCP)tag, green fluorescent protein (GFP) tag, small ubiquitin-like modifier(SUMO) tag, tandem affinity purification (TAP) tag, HaloTag, Nus-tag,Thioredoxin-tag, Fc-tag, CYD tag, HPC tag, TrpE tag, ubiquitin tag,VSV-G epitope tag, V5 tag, and a combination thereof; optionally,wherein the first or second affinity acceptor peptide comprises two ormore repeats of a tag sequence.

In some embodiments, the affinity acceptor peptide binding molecule isbiotin or an antibody specific to the affinity acceptor peptide. In someembodiments, the affinity acceptor peptide binding molecule bindsspecifically to an affinity molecule. In some embodiments, the affinitymolecule is streptavidin, NeutrAvidin, or a derivative thereof. In someembodiments, the affinity acceptor peptide binding molecule does notspecifically interact with an amino acid sequence of the recombinantclass I or class II HLA. In some embodiments, for two or more of therecombinant polynucleic acids: the sequence encoding the affinityacceptor tagged HLA is stably integrated into the genome of a cell. Insome embodiments, the sequence encoding β2 microglobulin or the sequenceencoding the HLA class II β-chain is connected to a sequence encoding asecond affinity acceptor peptide. In some embodiments, the secondaffinity acceptor peptide comprises an HA tag. In some embodiments, thesequence encoding β2 microglobulin or the sequence encoding the HLAclass II β-chain is connected to the sequence encoding the recombinantHLA and the affinity acceptor peptide by a linker.

In some embodiments, the linker comprises a polynucleic acid sequenceencoding a cleavable linker. In some embodiments, the cleavable linkeris a ribosomal skipping site or an internal ribosomal entry site (IRES)element. In some embodiments, the ribosomal skipping site or IRES iscleaved when expressed in the cells. In some embodiments, the ribosomalskipping site is selected from the group consisting of F2A, T2A, P2A,and E2A In some embodiments, the IRES element is selected from commoncellular or viral IRES sequences.

Provided herein is a composition comprising two or more isolatedpolypeptide molecules encoded by the polynucleic acid of a compositiondescribed herein. Provided herein is a composition comprising apopulation of cells comprising two or more polypeptide molecules encodedby the polynucleic acid of a composition described herein. Providedherein is a composition comprising a population of cells comprising acomposition described herein. Provided herein is a compositioncomprising a population of cells comprising one or more cells comprisinga composition described herein.

In some embodiments, the population of cells express one or moreendogenous class I or class II HLA alleles. In some embodiments, thepopulation of cells are engineered to lack one or more endogenous HLAclass I alleles. In some embodiments, the population of cells areengineered to lack endogenous HLA class I alleles. In some embodiments,the population of cells are engineered to lack one or more endogenousHLA class II alleles. In some embodiments, the population of cells areengineered to lack endogenous HLA class II alleles. In some embodiments,the population of cells are engineered to lack one or more endogenousHLA class I alleles and one or more endogenous HLA class II alleles. Insome embodiments, the population of cells is a population of low cellsurface HLA class I or class II expressing cells. In some embodiments,the composition is formulated using peptides or polynucleic acidsencoding peptides specific to an HLA type of a patient. Provided hereinis a method of making a cell comprising transducing or transfecting twoor more cells with the two or more polynucleic acids of a compositiondescribed herein.

Provided herein is a peptide identified according to a method describedherein. Provided herein is a method of inducing an anti-tumor responsein a mammal comprising administering to the mammal an effective amountof a polynucleic acid comprising a sequence of a peptide describedherein. Provided herein is a method of inducing an anti-tumor responsein a mammal comprising administering to the mammal an effective amountof a peptide comprising the sequence of a peptide described herein.Provided herein is a method of inducing an anti-tumor response in amammal comprising administering to the mammal a cell comprising apeptide comprising the sequence of a peptide described herein. Providedherein is a method of inducing an anti-tumor response in a mammalcomprising administering to the mammal a cell comprising an effectiveamount of a polynucleic acid comprising a sequence encoding a peptidecomprising the sequence of a peptide described herein. In someembodiments, the cell presents the peptide as an HLA-peptide complex.Provided herein is a method of for inducing an immune response in amammal comprising administering to the mammal an effective amount of apolynucleic acid comprising a sequence encoding a peptide describedherein. Provided herein is a method for inducing an immune response in amammal comprising administering to the mammal an effective amount of apeptide comprising the sequence of a peptide described herein. Providedherein is a method for inducing an immune response in a mammalcomprising administering to the mammal an effective amount of a cellcomprising a peptide comprising the sequence of a peptide describedherein. Provided herein is a method for inducing an immune response in amammal comprising administering to the mammal an effective amount of acell comprising a polynucleic acid comprising a sequence encoding apeptide comprising the sequence of a peptide described herein.

In some embodiments, the immune response is a T cell immune response. Insome embodiments, the immune response is a CD8 T cell response. In someembodiments, the immune response is a CD4 T cell response. In someembodiments, the immune response is humoral immune response.

Provided herein is a method for treating a mammal having a diseasecomprising administering to the mammal an effective amount of apolynucleic acid comprising a sequence encoding a peptide describedherein. Provided herein is a method for treating a mammal having adisease comprising administering to the mammal an effective amount of apeptide comprising the sequence of a peptide described herein. Providedherein is a method for treating a mammal having a disease comprisingadministering to the mammal an effective amount of a cell comprising apeptide comprising the sequence of a peptide described herein. Providedherein is a method for treating a mammal having a disease comprisingadministering to the mammal an effective amount of a cell comprising apolynucleic acid comprising a sequence encoding a peptide comprising thesequence of a peptide described herein. In some embodiments, the diseaseis cancer. In some embodiments, the disease is infection by aninfectious agent. In some embodiments, the infectious agent is apathogen, optionally a virus or bacteria, or a parasite.

In some embodiments, the virus is selected from the group consisting of:BK virus (BKV), Dengue viruses (DENV-1, DENV-2, DENV-3, DENV-4, DENV-5),cytomegalovirus (CMV), Hepatitis B virus (HBV), Hepatitis C virus (HCV),Epstein-Barr virus (EBV), an adenovirus, human immunodeficiency virus(HIV), human T-cell lymphotrophic virus (HTLV-1), an influenza virus,RSV, HPV, rabies, mumps rubella virus, poliovirus, yellow fever,hepatitis A, hepatitis B, Rotavirus, varicella virus, humanpapillomavirus (HPV), smallpox, zoster, and any combination thereof.

In some embodiments, the bacteria is selected from the group consistingof: Klebsiella spp., Tropheryma whipplei, Mycobacterium leprae,Mycobacterium lepromatosis, and Mycobacterium tuberculosis, typhoid,pneumococcal, meningococcal, haemophilus B, anthrax, tetanus toxoid,meningococcal group B, bcg, cholera, and any combination thereof.

In some embodiments, the parasite is a helminth or a protozoan. In someembodiments, the parasite is selected from the group consisting of:Leishmania spp., Plasmodium spp., Trypanosoma cruzi, Ascarislumbricoides, Trichuris trichiura, Necator americanus, Schistosoma spp.,and any combination thereof.

Provided herein is a method of enriching for immunogenic peptidescomprising: providing a population of cells comprising one or more cellsexpressing an affinity acceptor tagged HLA, wherein the affinityacceptor tagged HLA comprises an affinity acceptor peptide operativelylinked to a recombinant HLA encoded by a recombinant HLA allele; andenriching for HLA-peptide complexes comprising the affinity acceptortagged HLA. In some embodiments, the method further comprisesdetermining the sequence of immunogenic peptides isolated from theHLA-peptide complexes. In some embodiments, the determining comprisesusing LC-MS/MS.

Provided herein is a method of treating a disease or disorder in asubject, the method comprising administering to the subject an effectiveamount of a polynucleic acid comprising a sequence encoding a peptidedescribed herein. Provided herein is a method of treating a disease ordisorder in a subject, the method comprising administering to thesubject an effective amount of a peptide comprising the sequence of apeptide described herein. Provided herein is a method of treating adisease or disorder in a subject, the method comprising administering tothe subject an effective amount of a cell comprising a peptidecomprising the sequence of a peptide described herein. Provided hereinis a method of treating a disease or disorder in a subject, the methodcomprising administering to the subject a cell comprising an effectiveamount of a polynucleic acid comprising a sequence encoding a peptidecomprising the sequence of a peptide described herein.

Provided herein is a method of developing an therapeutic for a subjectwith a disease or condition comprising providing a population of cellsderived from a subject with a disease or condition, expressing in one ormore cells of the population of cells an affinity acceptor tagged classI or class II HLA allele by introducing into the one or more cells apolynucleic acid encoding a sequence comprising: a sequence encoding arecombinant class I or class II HLA allele operatively linked to asequence encoding an affinity acceptor peptide, thereby forming affinityacceptor tagged HLA-peptide complexes in the one or more cells;enriching and characterizing the affinity acceptor tagged HLA-peptidecomplexes; and, optionally, developing an therapeutic based on thecharacterization.

Provided herein is a method of identifying at least one subject specificimmunogenic antigen and preparing a subject-specific immunogeniccomposition that includes the at least one subject specific immunogenicantigen, wherein the subject has a disease and the at least one subjectspecific immunogenic antigen is specific to the subject and thesubject's disease, said method comprising: providing a population ofcells derived from a subject with a disease or condition, expressing inone or more cells of the population of cells from the subject, anaffinity acceptor tagged class I or class II HLA allele by introducinginto the one or more cells a polynucleic acid encoding a sequencecomprising: a sequence encoding a recombinant class I or class II HLAallele operatively linked to a sequence encoding an affinity acceptorpeptide, thereby forming affinity acceptor tagged HLA-peptide complexesin the one or more cells; enriching affinity acceptor tagged HLA-peptidecomplexes from the one or more cells; identifying an immunogenic peptidefrom the enriched affinity acceptor tagged HLA-peptide complexes that isspecific to the subject and the subject's disease; and formulating asubject-specific immunogenic composition based one or more of thesubject specific immunogenic peptides identified.

In some embodiments, the therapeutic or subject specific immunogeniccomposition comprises a peptide from the enriched affinity acceptortagged HLA-peptide complexes or a or a polynucleotide encoding thepolypeptide from the enriched affinity acceptor tagged HLA-peptidecomplexes. In some embodiments, the therapeutic or subject specificimmunogenic composition comprises a T cell expressing a T cell receptor(TCR) that specifically binds to the polypeptide from the enrichedaffinity acceptor tagged HLA-peptide complexes. In some embodiments, thesubject specific immunogenic composition comprises a chimeric antigenreceptor (CAR) T cell expressing a receptor that specifically binds tothe polypeptide from the enriched affinity acceptor tagged HLA-peptidecomplexes.

In some embodiments, the method further comprises administering anothertherapeutic agent, optionally, an immune checkpoint inhibitor to thesubject. In some embodiments, the method further comprises administeringan adjuvant, optionally, poly-ICLC to the subject.

In some embodiments, the disease or disorder is cancer. In someembodiments, the disease or disorder is an autoimmune disease. In someembodiments, the disease or disorder is an infection. In someembodiments, the infection is an infection by an infectious agent. Insome embodiments, the infectious agent is a pathogen, a virus, bacteria,or a parasite.

In some embodiments, the virus is selected from the group consisting of:BK virus (BKV), Dengue viruses (DENV-1, DENV-2, DENV-3, DENV-4, DENV-5),cytomegalovirus (CMV), Hepatitis B virus (HBV), Hepatitis C virus (HCV),Epstein-Barr virus (EBV), an adenovirus, human immunodeficiency virus(HIV), human T-cell lymphotrophic virus (HTLV-1), an influenza virus,RSV, HPV, rabies, mumps rubella virus, poliovirus, yellow fever,hepatitis A, hepatitis B, Rotavirus, varicella virus, humanpapillomavirus (HPV), smallpox, zoster, and any combination thereof.

In some embodiments, the bacteria is selected from the group consistingof: Klebsiella spp., Tropheryma whipplei, Mycobacterium leprae,Mycobacterium lepromatosis, and Mycobacterium tuberculosis, typhoid,pneumococcal, meningococcal, haemophilus B, anthrax, tetanus toxoid,meningococcal group B, bcg, cholera, and combinations thereof.

In some embodiments, the parasite is a helminth or a protozoan. In someembodiments, the parasite is selected from the group consisting of:Leishmania spp., Plasmodium spp., Trypanosoma cruzi, Ascarislumbricoides, Trichuris trichiura, Necator americanus, Schistosoma spp.,and any combination thereof.

Provided herein is a method of developing a therapeutic for a subjectwith a disease or condition comprising: providing a population of cells,wherein one or more cells of the population of cells comprise apolynucleic acid comprising a sequence encoding at least two affinityacceptor tagged class I or class II HLA alleles, wherein the sequenceencoding the at least two affinity acceptor tagged class I or class IIHLAs comprises a first recombinant sequence comprising a sequenceencoding a first class I or class II HLA allele operatively linked to asequence encoding a first affinity acceptor peptide; and a secondrecombinant sequence comprising a sequence encoding a second class I orclass II HLA allele operatively linked to a sequence encoding a secondaffinity acceptor peptide; expressing the at least two affinity acceptortagged HLAs in at least one cell of the one or more cells of thepopulation of cells, thereby forming affinity acceptor taggedHLA-peptide complexes in the at least one cell; enriching for theaffinity acceptor tagged HLA-peptide complexes; and identifying apeptide from the enriched affinity acceptor tagged HLA-peptidecomplexes; and formulating an immunogenic composition based one or moreof the peptides identified, wherein the first and the second recombinantclass I or class II HLA alleles are matched to an HLA haplotype of asubject. In some embodiments, the subject has a disease or condition.

In some embodiments, the first recombinant class I or class II HLAallele is different than the second recombinant class I or class II HLAallele. In some embodiments, the first affinity acceptor peptide is thesame as the second affinity acceptor peptide. In some embodiments, themethod comprises characterizing a peptide bound to the first and/orsecond affinity acceptor tagged HLA-peptide complexes from theenriching. In some embodiments, the at least two affinity acceptortagged class I or class II HLA alleles comprise at least 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or50 class I and/or class II HLA alleles. In some embodiments, the firstand/or the second affinity acceptor tagged HLA-peptide complexescomprise a transmembrane domain. In some embodiments, the first and/orthe second affinity acceptor tagged HLA-peptide complexes comprise anintracellular domain. In some embodiments, the first and/or the secondaffinity acceptor tagged HLA-peptide complexes are not excreted. In someembodiments, the first and/or the second affinity acceptor taggedHLA-peptide complexes incorporate into a cell membrane when expressed.In some embodiments, the first and/or the second affinity acceptortagged HLA-peptide complexes are not soluble affinity acceptor taggedHLA-peptide complexes.

In some embodiments, the method further comprises generating anHLA-allele specific peptide database. In some embodiments, the methodcomprises introducing one or more exogenous peptides to the populationof cells. In some embodiments, the introducing comprises contacting thepopulation of cells with the one or more exogenous peptides orexpressing the one or more exogenous peptides in the population ofcells. In some embodiments, the introducing comprises contacting thepopulation of cells with one or more nucleic acids encoding the one ormore exogenous peptides.

In some embodiments, the one or more nucleic acids encoding the one ormore peptides is DNA. In some embodiments, the one or more nucleic acidsencoding the one or more peptides is RNA, optionally wherein the RNA ismRNA.

In some embodiments, the enriching does not comprise use of a tetramerreagent. In some embodiments, the method comprises determining thesequence of a peptide or a portion thereof bound to the first and/or thesecond affinity acceptor tagged HLA-peptide complex from the enriching.In some embodiments, the determining comprises biochemical analysis,mass spectrometry analysis, MS analysis, MS/MS analysis, LC-MS/MSanalysis, or a combination thereof.

In some embodiments, the method comprises evaluating a binding affinityor stability of a peptide or a portion thereof bound to the first and/orthe second affinity acceptor tagged HLA-peptide complex from theenriching. In some embodiments, the method comprises determining whethera peptide or a portion thereof bound to the first and/or the secondaffinity acceptor tagged HLA-peptide complex from the enriching containsone or more mutations. In some embodiments, the method comprisesevaluating associations of peptides with HLA molecules in the firstand/or the second affinity acceptor tagged HLA-peptide complex.

In some embodiments, the method comprises expressing a library ofpeptides in the population of cells, thereby forming a library ofaffinity acceptor tagged HLA-peptide complexes. In some embodiments, themethod comprises contacting to the population of cells a library ofpeptides or a library of sequences encoding peptides, thereby forming alibrary of affinity acceptor tagged HLA-peptide complexes. In someembodiments, the library comprises a library of peptides associated witha disease or condition.

In some embodiments, the disease or condition is cancer or an infectionwith an infectious agent. In some embodiments, the method comprisesintroducing the infectious agent or portions thereof into one or morecells of the population of cells. In some embodiments, the methodcomprises characterizing one or more peptides from the first and/or thesecond HLA-peptide complexes, optionally wherein the peptides are fromone or more target proteins of the infectious agent. In someembodiments, the method comprises characterizing one or more regions ofthe peptides from the one or more target proteins of the infectiousagent. In some embodiments, the method comprises identifying peptidesfrom the first and/or the second HLA-peptide complexes derived from aninfectious agent.

In some embodiments, the population of cells is from a biological samplefrom a subject with a disease or condition. In some embodiments, thepopulation of cells is a cell line. In some embodiments, the populationof cells is a population of primary cells. In some embodiments, thepeptide from the first and/or the second affinity acceptor taggedHLA-peptide complex is capable of activating a T cell from a subjectwhen presented by an antigen presenting cell. In some embodiments, themethod comprises comparing HLA-peptide complexes from diseased cells toHLA-peptide complexes from non-diseased cells. In some embodiments, themethod further comprises isolating peptides from the first and/or thesecond affinity acceptor tagged HLA-peptide complexes before theidentifying. In some embodiments, the population of cells is apopulation of low cell surface HLA class I or class II expressing cells.

In some embodiments, the population of cells expresses one or moreendogenous HLA alleles. In some embodiments, the population of cellsexpresses the endogenous HLA alleles normally expressed by thepopulation of cells. In some embodiments, the population of cells is anengineered population of cells lacking one or more endogenous HLA classI alleles. In some embodiments, the population of cells is an engineeredpopulation of cells lacking endogenous HLA class I alleles. In someembodiments, the population of cells is an engineered population ofcells lacking one or more endogenous HLA class II alleles. In someembodiments, the population of cells is an engineered population ofcells lacking endogenous HLA class II alleles. In some embodiments, thepopulation of cells is an engineered population of cells lackingendogenous HLA class I alleles and endogenous HLA class II alleles. Insome embodiments, the population of cells is a knock-out of one or moreHLA class I alleles. In some embodiments, the population of cells is aknock-out of one or more HLA class II alleles. In some embodiments, thepopulation of cells is a knock-out of all HLA class I alleles. In someembodiments, the population of cells is a knock-out of all HLA class IIalleles. In some embodiments, the population of cells is a knock-out ofall HLA class I alleles and a knock-out of all HLA class II alleles. Insome embodiments, the sequence encoding the at least two affinityacceptor tagged class I or class II HLA alleles encodes a class I HLA.In some embodiments, the class I HLA is selected from the groupconsisting of HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, and HLA-G. In someembodiments, the first recombinant class I or class II HLA allele is afirst class I HLA allele and the second recombinant class I or class IIHLA allele is a second class I HLA allele. In some embodiments, thesequence encoding the at least two affinity acceptor tagged class I orclass II HLA alleles encodes a class II HLA. In some embodiments, theclass II HLA is selected from the group consisting of HLA-DR, HLA-DQ,and HLA-DP. In some embodiments, the class II HLA comprises a HLA classII α-chain, a HLA class II β-chain, or a combination thereof. In someembodiments, the first recombinant class I or class II HLA allele is afirst class II HLA allele and the second recombinant class I or class IIHLA allele is a second class II HLA allele.

In some embodiments, the first sequence and the second sequence are eachoperatively linked. In some embodiments, the first sequence and thesecond sequence are comprised on different polynucleotide molecules. Insome embodiments, the sequence encoding the first and/or second affinityacceptor peptide is operatively linked to a sequence that encodes anextracellular portion of the first and/or second class I or class II HLAallele. In some embodiments, the first and/or second encoded affinityacceptor peptide is expressed extracellularly. In some embodiments, thesequence encoding the first and/or second affinity acceptor peptide isoperatively linked to the N-terminus of the sequence encoding the firstand/or second class I or class II HLA allele. In some embodiments, thesequence encoding the first and/or second affinity acceptor peptide isoperatively linked to a sequence that encodes an intracellular portionof the first and/or second class I or class II HLA allele. In someembodiments, the encoded first and/or second affinity acceptor peptideis expressed intracellularly. In some embodiments, the sequence encodingthe first and/or second affinity acceptor peptide is operatively linkedto the C-terminus of the sequence encoding the first and/or second classI or class II HLA allele. In some embodiments, the sequence encoding thefirst and/or second affinity acceptor peptide is operatively linked tothe sequence encoding the first and/or second class I or class II HLAallele by a linker.

In some embodiments, enriching comprises enriching for intact cellsexpressing the first and/or second affinity acceptor tagged HLA-peptidecomplexes. In some embodiments, the method does not comprise lysing thecells before enriching. In some embodiments, the method furthercomprises lysing the one or more cells before enriching. In someembodiments, enriching comprises contacting an affinity acceptor peptidebinding molecule to the first and/or second affinity acceptor taggedHLA-peptide complexes, wherein the affinity acceptor peptide bindingmolecule binds specifically to the first and/or second affinity acceptorpeptide.

In some embodiments, the first and/or second affinity acceptor peptidecomprises a tag sequence comprising a biotin acceptor peptide (BAP),poly-histidine tag, poly-histidine-glycine tag, poly-arginine tag,poly-aspartate tag, poly-cysteine tag, poly-phenylalanine, c-myc tag,Herpes simplex virus glycoprotein D (gD) tag, FLAG tag, KT3 epitope tag,tubulin epitope tag, T7 gene 10 protein peptide tag, streptavidin tag,streptavidin binding peptide (SPB) tag, Strep-tag, Strep-tag II,albumin-binding protein (ABP) tag, alkaline phosphatase (AP) tag,bluetongue virus tag (B-tag), calmodulin binding peptide (CBP) tag,chloramphenicol acetyl transferase (CAT) tag, choline-binding domain(CBD) tag, chitin binding domain (CBD) tag, cellulose binding domain(CBP) tag, dihydrofolate reductase (DHFR) tag, galactose-binding protein(GBP) tag, maltose binding protein (MBP), glutathione-S-transferase(GST), Glu-Glu (EE) tag, human influenza hemagglutinin (HA) tag,horseradish peroxidase (HRP) tag, NE-tag, HSV tag, ketosteroid isomerase(KSI) tag, KT3 tag, LacZ tag, luciferase tag, NusA tag, PDZ domain tag,AviTag, Calmodulin-tag, E-tag, S-tag, SBP-tag, Softag 1, Softag 3, TCtag, VSV-tag, Xpress tag, Isopeptag, SpyTag, SnoopTag, Profinity eXacttag, Protein C tag, S1-tag, S-tag, biotin-carboxy carrier protein (BCCP)tag, green fluorescent protein (GFP) tag, small ubiquitin-like modifier(SUMO) tag, tandem affinity purification (TAP) tag, HaloTag, Nus-tag,Thioredoxin-tag, Fc-tag, CYD tag, HPC tag, TrpE tag, ubiquitin tag,VSV-G epitope tag, V5 tag, or a combination thereof; optionally, whereinthe first and/or second affinity acceptor peptide comprises two or morerepeats of a tag sequence.

In some embodiments, the affinity acceptor peptide binding molecule isbiotin or an antibody specific to the first and/or second affinityacceptor peptide. In some embodiments, the enriching comprisescontacting an affinity molecule to the first and/or second affinityacceptor tagged HLA-peptide complexes, wherein the affinity moleculebinds specifically to the affinity acceptor peptide binding molecule. Insome embodiments, the affinity molecule is streptavidin, NeutrAvidin, ora derivative thereof. In some embodiments, enriching comprisesimmunoprecipitating the first and/or second affinity acceptor taggedHLA-peptide complexes.

In some embodiments, the affinity acceptor peptide binding molecule isattached to a solid surface. In some embodiments, the affinity moleculeis attached to a solid surface. In some embodiments, the solid surfaceis a bead.

In some embodiments, enriching comprises immunoprecipitating the firstand/or second affinity acceptor tagged HLA-peptide complexes with anaffinity acceptor peptide binding molecule that binds specifically tothe first and/or second affinity acceptor peptide. In some embodiments,the affinity acceptor peptide binding molecule does not specificallyinteract with the amino acid sequence of the encoded first and/or secondclass I or class II HLA. In some embodiments, enriching comprisescontacting an affinity molecule specific to an extracellular portion ofthe first and/or second class I or class II HLA allele. In someembodiments, enriching comprises contacting an affinity moleculespecific to an N-terminal portion of the first and/or second class I orclass II HLA allele.

In some embodiments, providing comprises contacting the population ofcells with the polynucleic acid. In some embodiments, contactingcomprises transfecting or transducing. In some embodiments, providingcomprises contacting the population of cells with a vector comprisingthe polynucleic acid. In some embodiments, the vector is a viral vector.In some embodiments, the polynucleic acid is stably integrated into thegenome of the population of cells.

In some embodiments, the sequence encoding the first and/or second classI or class II HLA comprises a sequence encoding a HLA class I α-chain.In some embodiments, the first recombinant class I or class II HLAallele is a first HLA class I α-chain and the second recombinant class Ior class II HLA allele is a second HLA class I α-chain.

In some embodiments, the method further comprises expressing a sequenceencoding β2 microglobulin in the one or more cells. In some embodiments,the sequence encoding β2 microglobulin is connected to the sequenceencoding the first and/or second class I or class II HLA. In someembodiments, the sequence encoding β2 microglobulin is connected to thesequence encoding the first and/or second class I or class II HLA by alinker. In some embodiments, the sequence encoding β2 microglobulin isconnected to a sequence encoding a third affinity acceptor peptide.

In some embodiments, the third affinity acceptor peptide is differentthan the first and/or second affinity acceptor peptide. In someembodiments, the sequence encoding the first and/or second class I orclass II HLA comprises a sequence encoding a HLA class II α-chain and/ora HLA class II β-chain. In some embodiments, the sequence encoding thefirst and/or second class I or class II HLA comprises a sequenceencoding a first HLA class II α-chain and a second HLA class II α-chain.In some embodiments, the method further comprises expressing a sequenceencoding a HLA class II β-chain in the one or more cells. In someembodiments, the sequence encoding a first HLA class II α-chain and asecond HLA class II α-chain HLA is connected to the sequence encodingthe HLA class II β-chain. In some embodiments, the sequence encoding thefirst and/or second class I or class II HLA comprises a sequenceencoding a first HLA class II β-chain and a second HLA class II β-chain.

In some embodiments, the method further comprises expressing a sequenceencoding a HLA class II α-chain in the one or more cells. In someembodiments, the sequence encoding a first HLA class II β-chain and asecond HLA class II β-chain is connected to the sequence encoding theHLA class II α-chain by a linker. In some embodiments, the sequenceencoding the HLA class II β-chain or the HLA class II α-chain isconnected to a sequence encoding a third affinity acceptor peptide. Insome embodiments, the third affinity acceptor peptide is different thanthe first and/or second affinity acceptor peptide.

In some embodiments, the third affinity acceptor peptide is differentthan the first affinity acceptor peptide and is selected from the groupconsisting of biotin acceptor peptide (BAP), poly-histidine tag,poly-histidine-glycine tag, poly-arginine tag, poly-aspartate tag,poly-cysteine tag, poly-phenylalanine, c-myc tag, Herpes simplex virusglycoprotein D (gD) tag, FLAG tag, KT3 epitope tag, tubulin epitope tag,T7 gene 10 protein peptide tag, streptavidin tag, streptavidin bindingpeptide (SPB) tag, Strep-tag, Strep-tag II, albumin-binding protein(ABP) tag, alkaline phosphatase (AP) tag, bluetongue virus tag (B-tag),calmodulin binding peptide (CBP) tag, chloramphenicol acetyl transferase(CAT) tag, choline-binding domain (CBD) tag, chitin binding domain (CBD)tag, cellulose binding domain (CBP) tag, dihydrofolate reductase (DHFR)tag, galactose-binding protein (GBP) tag, maltose binding protein (MBP),glutathione-S-transferase (GST), Glu-Glu (EE) tag, human influenzahemagglutinin (HA) tag, horseradish peroxidase (HRP) tag, NE-tag, HSVtag, ketosteroid isomerase (KSI) tag, KT3 tag, LacZ tag, luciferase tag,NusA tag, PDZ domain tag, AviTag, Calmodulin-tag, E-tag, S-tag, SBP-tag,Softag 1, Softag 3, TC tag, VSV-tag, Xpress tag, Isopeptag, SpyTag,SnoopTag, Profinity eXact tag, Protein C tag, S1-tag, S-tag,biotin-carboxy carrier protein (BCCP) tag, green fluorescent protein(GFP) tag, small ubiquitin-like modifier (SUMO) tag, tandem affinitypurification (TAP) tag, HaloTag, Nus-tag, Thioredoxin-tag, Fc-tag, CYDtag, HPC tag, TrpE tag, ubiquitin tag, VSV-G epitope tag, V5 tag, and acombination thereof; optionally, wherein the first or second affinityacceptor peptide comprises two or more repeats of a tag sequence.

In some embodiments, the linker comprises a polynucleic acid sequenceencoding a cleavable linker. In some embodiments, the cleavable linkeris a ribosomal skipping site or an internal ribosomal entry site (IRES)element. In some embodiments, the ribosomal skipping site or IRES iscleaved when expressed in the cells. In some embodiments, the ribosomalskipping site is selected from the group consisting of F2A, T2A, P2A,and E2A. In some embodiments, the IRES element is selected from commoncellular or viral IRES sequences.

In some embodiments, the method comprises performing biochemicalanalysis or mass spectrometry, such as tandem mass spectrometry. In someembodiments, the method comprises obtaining a peptide sequence thatcorresponds to an MS/MS spectra of one or more peptides isolated fromthe enriched affinity acceptor tagged HLA-peptide complexes from apeptide database; wherein one or more sequences obtained identifies thesequence of the one or more peptides.

In some embodiments, the population of cells is a cell line selectedfrom HEK293T, expi293, HeLa, A375, 721.221, JEG-3, K562, Jurkat, andTHP1. In some embodiments, the cell line is treated with one or morecytokines, checkpoint inhibitors, epigenetically-active drugs, IFN-γ, ora combination thereof. In some embodiments, the population of cellscomprises at least 10⁵ cells, at least 10⁶ cells or at least 10⁷ cells.In some embodiments, the population of cells is a population ofdendritic cells, macrophages, cancer cells or B-cells. In someembodiments, the population of cells comprises tumor cells.

In some embodiments, the population of cells is contacted with an agentprior to isolating the first and/or second HLA-peptide complexes fromthe one or more cells. In some embodiments, the agent is an inflammatorycytokine, a chemical agent, an adjuvant, a therapeutic agent orradiation.

In some embodiments, the first and or second HLA allele is a mutated HLAallele. In some embodiments, the sequence encoding the first and orsecond HLA allele comprises a barcode sequence. In some embodiments, themethod further comprises assaying for expression of the first and/orsecond affinity acceptor tagged class I or class II HLA allele.

In some embodiments, the assaying comprises sequencing the first and/orsecond affinity acceptor tagged class I or class II HLA allele,detecting RNA encoding the first and/or second affinity acceptor taggedclass I or class II HLA allele RNA, detecting the first and/or secondaffinity acceptor tagged class I or class II HLA allele protein, or acombination thereof. In some embodiments, the first and second affinityacceptor tagged class I or class II HLA allele comprises a uniquebarcode sequence. In some embodiments, the first sequence and the secondsequence comprise a unique barcode sequence.

1.-76. (canceled)
 77. A method of training an MHC-peptide presentationprediction algorithm implemented in a computer processor, the methodcomprising: (a) contacting a plurality of training peptides with aplurality of training cells in vitro, wherein each training cell of theplurality of training cells expresses a recombinant MHC protein thatincorporates into a cell membrane of a training cell; and wherein (i)the training cell does not express an endogenous MHC protein, or (ii)the recombinant MHC protein is operably linked to an affinity acceptorpeptide sequence; and (b) immunoprecipitating a recombinant MHC proteinof plurality of training cells; (c) identifying training peptides boundto the recombinant MHC protein immunoprecipitated in (b) by massspectrometry; and (d) inputting as a variable into the presentationprediction algorithm: (i) amino acid sequence information of thetraining peptides identified by mass spectrometry, and (ii) informationof the recombinant MHC protein immunoprecipitated in (b), therebytraining the MHC-peptide presentation prediction algorithm.
 78. Themethod of claim 77, wherein the recombinant MHC protein is an WIC classII protein.
 79. The method of claim 77, wherein the plurality oftraining cells do not express an endogenous MHC protein.
 80. The methodof claim 79, wherein immunoprecipitating comprises immunoprecipitatingwith an affinity molecule that binds to the affinity acceptor peptidesequence wherein the affinity molecule is streptavidin, NeutrAvidin. 81.The method of claim 79, wherein the plurality of training cells expressa single recombinant MHC protein.
 82. The method of claim 77, whereinthe plurality of training cells is an antigen presenting cell line. 83.The method of claim 77, wherein each training peptide of plurality oftraining peptides is an endogenous peptide of the training cells. 84.The method of claim 77, wherein the recombinant MHC protein is operablylinked to an affinity acceptor peptide sequence.
 85. The method of claim84, wherein the affinity acceptor peptide sequence is operably linked toan extracellular portion of the recombinant MHC protein.
 86. The methodof claim 85, wherein immunoprecipitating comprises immunoprecipitatingintact training cells.
 87. The method of claim 84, whereinimmunoprecipitating comprises immunoprecipitating with an affinitymolecule that binds to the affinity acceptor peptide sequence.
 88. Themethod of claim 87, wherein the affinity acceptor peptide sequence isBiotin Acceptor Protein (BAP) sequence.
 89. The method of claim 88,wherein the affinity molecule is streptavidin, NeutrAvidin, or aderivative thereof.
 90. The method of claim 84, wherein the plurality oftraining cells expresses: (a) a first recombinant MHC protein comprisinga first MHC molecule and a first affinity acceptor peptide; and (b) asecond recombinant MHC protein comprising a second MHC molecule and asecond affinity acceptor peptide, wherein the first MHC molecule and thesecond MHC molecule are non-identical; and wherein the first affinityacceptor peptide and the second affinity acceptor peptide arenonidentical.
 91. The method of claim 77, wherein identifying furthercomprises performing a sequencing analysis, a biochemical analysis orcombination thereof.
 92. The method of claim 77, wherein the methodfurther comprises inputting into the presentation prediction algorithminformation of one or more variables selected from the group consistingof amino acid physical properties, peptide physical properties,expression level of the source protein of the peptide within a cell,protein stability, protein translation rate, ubiquitination sites,protein degradation rate, translational efficiencies from ribosomalprofiling, protein cleavability, protein localization, motifs of hostprotein that facilitate TAP transport, host protein is subject toautophagy, motifs that favor ribosomal stalling, protein features thatfavor non-sense mediated degradation (NMD).
 93. The method of claim 77,wherein the method comprises generating an HLA-allele specific peptidedatabase.
 94. The method of claim 77, wherein the method furthercomprises training the MHC-peptide presentation prediction algorithmwith information from an HLA-allele specific peptide database.
 95. Themethod of claim 77, further comprising selecting one or more peptidesfrom a plurality of candidate peptide sequences expressed by cancercells of a single subject using the MHC-peptide presentation predictionalgorithm implemented in a computer processor that has been trainedaccording to claim 77, wherein the one or more peptides from theplurality of candidate peptide sequences is predicted to bind to an MEWprotein expressed by a cell of the single subject by the MHC-peptidepresentation prediction algorithm.
 96. The method of claim 95, furthercomprising administering to a subject in need thereof, a pharmaceuticalcomposition comprising the one or more peptides selected using theMHC-peptide presentation prediction algorithm, or a nucleic acidsequence encoding the one or more peptides.